Mass Sppyectrometry - Prince of Songkla Universityhost.pharmacy.psu.ac.th/tvimon/mass-adv.pdf ·...

Mass Spectrometryp y

Pharmaceutical Chemistry f D D l pm ntfor Drug Development565-601 (2 hr)

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67 Analysis of biomoleculesbiomolecules

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Block diagram of features of a gtypical mass spectrometer

Mass analyzerMass analyzer

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Ionization Methods1. Gas-Phase Ionization Methods

1.1 Electron Impact Ionization (EI)1.2 Chemical Ionization (CI)

2. Desorption Ionization Methods2.1 Field Desorption Ionization (FD) 2.2 Fast Atom Bombardment Ionization (FAB).. 10 & 20 kDa

Pl (P )2.3 Plasma Desorption Ionization (PD).. 45 kDa2.4 Laser Desorption Ionization (LD)

3 E ti I i ti M th d3. Evaporative Ionization Methods3.1 Thermospray Mass Spectrometry3 2 El t M S t t (ES)

43.2 Electrospray Mass Spectrometry (ES).. 100 kDa

Summary of Ionization Methods

ionization methods that are used most often to study the peptides and proteins 5

ionization methods that are used most often to study the peptides and proteins through mass spectrometry are ESI and MALDI.

Electron Impact lonization (EI)

most widely used methodVapor phase sample molecules are bombarded with high-energy bombarded with high energy electrons (generally 70 eV), which eject an electron from a sample eject an electron from a sample molecule to produce a radical cation, k th l l iknown as the molecular ion

61.1


ionization potential of typical organic compounds is generally less than 15 eV, the p g ybombarding electrons impart 50 eV (or more) of excess energy to the newly created molecular i hi h i di i t d i t b th b ki ion, which is dissipated in part by the breaking of covalent bonds, which have bond strengths between 3 and 10 eVbetween 3 and 10 eV.

Bond breaking is usually extensivef t ti i l ” di t bl ” d fragmentation process is also ”predictable” and is the source of the powerful structure elucidation potential of mass spectrometry

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elucidation potential of mass spectrometry.1.1


excess energy imparted to the molecular ion is too great, which leads to a mass spectrum with no pdiscernible molecular ion.

81.1

Chemical lonization (CI)

“soft ionization”In CI, sample molecules (in the vapor phase) are not subjected to p ) jbombardment by high energy electrons. Reagent gas (usually methane Reagent gas (usually methane,

isobutane, ammonia, but others are used) is introduced into the sourceused) is introduced into the source,and ionized.

91.2


Sample molecules collide with ionized reagent gas molecules (CH + C H + etc) in reagent gas molecules (CH5 , C4H9 , etc) in the relatively high-pressure CI source, and undergo secondary ionization and undergo secondary ionization

by proton transfer producing an [M + 1]+ ion, by electrophilic addition producing [M + 15]+ by electrophilic addition producing [M + 15] , [M + 24]+, [M + 43]+ or [M + 18]+ (with NH4

+) ions, or by charge exchange (rare) producing a [M]+

ion.

101.2


Chemical ionization spectra sometimes have prominent [M 1]+ ions because of have prominent [M – 1]+ ions because of hydride abstraction.

Th t f d t th The excess energy transfered to the sample molecules during the ionization h s is sm ll ll l ss th 5 V phase is small, generally less than 5 eV,

so much less fragmentation takes placeplace.less information on structure.

111.2

EI d CI t f 3 4 di th t hEI and CI mass spectra of 3,4-dimethoxyacetophenone.

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m/z 209 ([M + 29]+ or M + C2H5+) and

m/z 221 ([M + 41]+ or M + C3H5+) m/z 221 ([M + 41] or M + C3H5 )

are a result of electrophilic addition of carbocations and are very useful in indentifing the molecular ion molecular ion.

The excess methane carrier gas is ionized by electron impact to the primary ions CH4

+ and p p y 4CH3+.

These react with the excess methane to give secondary ions secondary ions.

131.2


energy content of the various secondary ions decrease in the order: CH5

+ > t-C4H9+ > NH4

+decrease in the order: CH5 > t C4H9 > NH4 .choice of reagent gas, we can control the tendency of the CI produced [M + 1]+ ion to tendency of the CI produced [M 1] ion to fragment.CI not useful

for peak matching (either manually or by computer)

for structure elucidation;its main use is for the detection of molecular ionsand hence molecular weights.

141.2

Desorption Ionization Methods

sample molecules are emitted directly from a condensed phase into the vapor phase as ions. p pThe primary use is for

l r n nv l til r i nic c mp undslarge, nonvolatile, or ionic compounds.The resulting spectra are often g pcomplicated by abundant matrix ions.

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Field Desorption lonization (FD)

the sample is applied to a metal emitter on the surface of which is found carbon microneedles. The microneedles activate the surface, which is maintained at the accelerating voltage and functions as the anode.

Very high voltage gradients at the tips of the needles Very high voltage gradients at the tips of the needles remove an electron from the sample, and the resulting cation is repelled away from the emitter.The ions generated have little excess energy so there is The ions generated have little excess energy so there is minimal fragmentation, i.e., the molecular ion is usually the only significant ion seen. For example with cholesten-5-ene-3 16 22 26-tetrol For example with cholesten 5 ene 3,16,22,26 tetrol

EI and CI do not see a molecular ion FD mass spectrum shows predominately the molecular ion with virtually no fragmentation.

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y g

2.1

EI

CICI

FD

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Fast Atom Bombardment Ionization (FAB)

uses high-energy xenon or argon atoms (6 10 keV) to bombard samples dissolved (6-10 keV) to bombard samples dissolved in a liquid of low vapor pressure (e.g. glycerol)glycerol).

The matrix protects the sample from excessive radiation damage. excessive radiation damage. A related method, liquid secondary ionization mass spectrometry LSIMS is ionization mass spectrometry, LSIMS, is similar except that it uses somewhat more energetic cesium ions (10-30 keV).

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g ( )2.2


Formpositive ions by cation attachmentpositive ions by cation attachment

[M + 1]+ or [M + 23, Na]+) [M 3, Na] )

negative ions: by deprotonation [M -1]+

both types of ions are usually singly chargedFAB is used primarily with large nonvolatile

l l l l d l l molecules, particularly to determine molecular weight

192.2

Fast Atom Bombardment Ionization (FAB)For most classes of compounds, the rest of the spectrum is less useful, partially because the lower

b d f i d d b mass ranges may be composed of ions produced by the matrix itself.for certain classes of compounds that are composed for certain classes of compounds that are composed of "building blocks," such as polysaccharides and peptides,

some structural information may be obtained because fragmentation usually occurs at the glycosidic and peptide bonds respectivelyglycosidic and peptide bonds, respectively,thereby affording a method of sequencing these classes of compounds.

202.2


The upper mass limit for FAB (and LSIMS) ionization is between 10 and 20 kDa, and FAB is really most useful up to about 6 kDa useful up to about 6 kDa. FAB is seen most often with double focusing magnetic sector instruments where it has a resolution of about 0 3 l h i 0.3 mlz over the entire mass range; FAB can, however, be used with most types of mass analyzers analyzers.

The biggest drawback to using FAB is that the spectrum always shows a high level of matrixgenerated ions which limit sensitivity and which may generated ions, which limit sensitivity and which may obscure important fragment ions.

212.2

Plasma Desorption Ionization p(PD)

exclusively with a time of flight (TOF) mass analyzeranalyzerThe fission products from Californium 252 (252Cf), with energies in the range of 80-100 MeV,

s d t b mb d nd i ni th s mpl are used to bombard and ionize the sample. Each time a 252Cf splits, two particles are produced moving in opposite directionsproduced moving in opposite directions.

One of the particles hits a triggering detector and signals a start time.

The other particle strikes the sample matrix ejecting The other particle strikes the sample matrix ejecting some sample ions into a time of flight mass spectrometer (TOF-MS).

222.3

Plasma Desorption Ionization p(PD)

The sample ions are most often released as singly, doubly, or triply protonated moieties. singly, doubly, or triply protonated moieties. These ions are of fairly low energy so that structurally useful fragmentation is rarely b d d f l h id d

y g yobserved and, for polysaccharides and polypeptides, sequencing information is not availableavailable.The mass accuracy of the method is limited by the TOFMS.

The technique is useful on compounds with MWup to at least 45 kDa.

232.3

Laser Desorption Ionization p(LD)

A pulsed laser beam can be used to ionize samples for mass spectrometry. p p yBecause this method of ionization is pulsed, it must be used with either a time of flight or a Fourier transform mass spectrometerFourier transform mass spectrometerTwo types of lasers have found widespread use:

CO2 laser, which emits radiation in the far infrared regionregionfrequency-quadrupled neodymium/yttrium aluminum garnet (Nd/YAG) laser, which emits radiation in the UV region at 266 nm region at 266 nm.

Without matrix assistance. the method is limited to low MW molecules (< 2kDa).

242.4

Laser Desorption Ionization p(LD)

The power of the method is greatly enhanced by using matrix assistance (matrix assisted laser desorption ionization, or MALDI)MALDI).

MALDI is based on the bombardment of sample molecules with a laser light to bring about sample ionisation.

The sample is pre mixed with a highly absorbing matrix compound The sample is pre-mixed with a highly absorbing matrix compound for the most consistent and reliable results, and a low concentration of sample to matrix works best.

The matrix transforms the laser energy into excitation energyThe matrix transforms the laser energy into excitation energyfor the sample, which leads to sputtering of analyte and matrix ions from the surface of the mixture.

In this way energy transfer is efficient and also the analyzed n th way n rgy tran f r ff c nt an a th ana yz molecules are spared excessive direct energy that may otherwise cause decomposition.

Most commercially available MALDI mass spectrometers now have l d it l f l th 337

25a pulsed nitrogen laser of wavelength 337 nm.

2.4

The sample to be analysed is dissolved in an appropriate The sample to be analysed is dissolved in an appropriate volatile solvent, usually with a trace of trifluoroaceticacid if positive ionisation is being used, at a concentration of ca 10 pmol/µL and an aliquot (1 2 µL) concentration of ca. 10 pmol/µL and an aliquot (1-2 µL) of this removed and mixed with an equal volume of a solution containing a vast excess of a matrix.

A f d i i bl f iA range of compounds is suitable for use as matrices: sinapinic acid is a common one for protein analysisalpha-cyano-4-hydroxycinnamic acid is often used for p y y y m f fpeptide analysis.

An aliquot (1-2 µL) of the final solution is applied to thesample target which is allowed to dry prior to insertion sample target which is allowed to dry prior to insertion into the high vacuum of the mass spectrometer.

The laser is fired, the energy arriving at the sample/matrix surface optimised and data accumulated

26sample/matrix surface optimised, and data accumulated until a m/z spectrum of reasonable intensity has been amassed.

matrix assisted laser desorption pionization (MALDI)

The time-of-flight analyser separates ions according to their m/z ratios by measuring the time g y git takes for ions to travel through a field free region known as the flight, or drift, tube. The heavier ions are slower than the lighter ones. gThe m/z scale of the mass spectrometer is calibrated with a known sample that can either be analysed independently (external calibration) or analysed independently (external calibration) or pre-mixed with the sample and matrix (internal calibration).

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Simplified schematic of MALDI-TOF Simplified schematic of MALDI-TOF mass spectrometry (linear mode)

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MALDI is also a "soft" ionisation method and so results predominantly in the generation of singly charged molecular-related ions regardless of the molecular g fmass, hence the spectra are relatively easy to interpret.

Fragmentation of the sample ions does not usually Fragmentation of the sample ions does not usually occur.

In positive ionisation mode the protonated molecular i (M H)+ s ll th d i t s i s lth h ions (M+H)+ are usually the dominant species, although they can be accompanied by salt adducts, a trace of the doubly charged molecular ion at approximately half th / l d/ t f di i i t the m/z value, and/or a trace of a dimeric species at approximately twice the m/z value.

Positive ionisation is used in general for protein and 29

g f ppeptide analyses.


In negative ionisation mode the deprotonated molecular ions (M H)- are deprotonated molecular ions (M-H) are usually the most abundant species, accompanied by some salt adducts and accompanied by some salt adducts and possibly traces of dimeric or doubly charged materials. gNegative ionisation can be used for the analysis of oligonucleotides and y goligosaccharides.

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The mass accuracy is low when used with a TOF MS but very high resolution can a TOF-MS, but very high resolution can be obtained with a FT-MS.

As with other matrix assisted methods As with other matrix-assisted methods, MALDI suffers from background interference from the matrix material interference from the matrix material, which is further exacerbated by matrix adduction. Thus, the assignment of a molecular ion of an unknown compound can be uncertain.

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p2.4

Evaporative Ionization Methods

There are two important methods in which ions or less often neutral compounds in ions or, less often, neutral compounds in solution (often containing formic acid)

have their solvent molecules stripped by have their solvent molecules stripped by evaporation,

with simultaneous ionization leaving behind the ions for mass analysis.

Coupled with liquid chromatography p q g p yinstrumentation, these methods have become immensely popular.

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Thermospray Mass Spectrometry

a solution of the sample is introduced into the mass spectrometer by means of a heated capillary tube. p y p yThe tube nebulizes and partially vaporizes the solvent forming a stream of fine droplets, which enter the ion source enter the ion source.

When the solvent completely evaporates, the sample ions can be mass analyzed.

h h d h dl h h fl d b ff This method can handle high flow rates and buffers; it was an early solution to interfacing mass spectrometers with aqueous liquid chromatography. p q q g p yThe method has largely been supplanted by electrospray.

333.1

Electrospray (ES) Mass p y ( )Spectrometry

Electrospray ionization (ESI) is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to overcomes the propensity of these molecules to fragment when ionized.

The development of ESI for the analysis of biological l l d d i h h ib i f h

p y gmacromolecules was rewarded with the attribution of the Nobel Prize in Chemistry to John Bennett Fenn in 2002.Mass spectrometry using ESI is called Mass spectrometry using ESI is called

electrospray ionization mass spectrometry (ESI-MS)or, less commonly, electrospray mass spectrometry (ES-MS)MS).

343.2

the Nobel prize in Chemistry (2002) for John B. Fenn, Professor at the Virginia Commonwealth University, for his contributions to electrospray ionisation (ESI)to electrospray ionisation (ESI), and to Koichi Tanaka, an engineer at Shimadzu Corp Japan for the development of matrix-Corp., Japan, for the development of matrix-assisted laser desorption ionisation (MALDI),sharing the prize with Kurt Wuthrich at ETHsharing the prize with Kurt Wuthrich at ETH Zurich, Switzerland, for his contributions to protein 3D structure elucidation by NMR.

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Electrospray (ES) Mass Electrospray (ES) Mass Spectrometry

ES ion source is operated at or near atmospheric pressure and, thus is also called atmospheric pressure p , p pionization or API.

The sample in solution (usually a polar, volatile solvent) enters the ion source through a stainless steel enters the ion source through a stainless steel capillary, which is surrounded by a co-axial flow of nitrogen called the nebulizing gas.

The tip f the capillary is maintained at a hi h The tip of the capillary is maintained at a high potential with respect to a counter-electrode.The potential difference produces a field gradient of

k / p p g

up to 5 kV/cm. As the solution exits the capillary, an aerosol of charged droplets forms The flow of nebulizing gas

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charged droplets forms. The flow of nebulizing gas directs the effluent toward the mass spectrometer.

3.2


Droplets in the aerosol shrink as the solvent evaporates thereby solvent evaporates, thereby concentrating the charged sample ions.

When the electrostatic repulsion among p gthe charged sample ions reaches a critical point, the droplet undergoes a so-called “Coulombic explosion” which releases the Coulombic explosion which releases the sample ions into the vapor phase.

The vapor phase ions are focused with a The vapor phase ions are focused with a number of sampling orifices into the mass analyzer.

373.2

A diagram showing the evaporation of solvent leading to individual ions in an electrosprayleading to individual ions in an electrosprayinstrument.

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39

Standard electrospray p yionisation source

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The electrospray ionisation p yprocess

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for compounds that have multiple charge bearing sites.

With proteins, for example, ions with multiple charges are formed.

Since the mass spectrometer measures mass to Since the mass spectrometer measures mass to charge ratio (m/z) rather than mass directly, these multiply charged ions are recorded at apparent mass values of 1/2 1/3 1/n of their apparent mass values of 1/2, 1/3, … 1/n of their actual masses, where n is the number of charges (z).

Large proteins can have 40 or more charges so Large proteins can have 40 or more charges so that molecules of up to 100 kDa can be detectedin the range of conventional quadrupole, ion trap. or magnetic sector mass spectrometers

42or magnetic sector mass spectrometers.

3.2


The appearance of the spectrum is a series of peaks increasing in mass, which of pea s ncreas ng n mass, wh ch correspond to pseudo molecular ionspossessing sequentially one less proton and p g q y ptherefore one less charge.

Determination of the actual mass of the ion requires that the charge of the ion be known.

If two peaks, which differ by a single charge, can b id tifi d th l l ti i d d t i l be identified, the calculation is reduced to simple algebra.

433.2

The El and ES mass spectra of lactose.

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The El and ES mass spectra of lactose.

The EI mass spectrum of lactose is completely useless because completely useless because

lactose has low vapor pressure,

it is thermally labile, and ythe spectrum shows no characteristic peaks.

The ES mass spectrum shows k l l k / 4 d a weak molecular ion peak at m/z 342 and

a characteristic [M + 23]+, the molecular ion peak plus sodium peak plus sodium. Because sodium ions are ubiquitous in aqueous solution, these sodium adducts are very common

45common.

3.2

ES mass spectrum for the tetra-peptidetetra peptide comprised of valine lycine serine and lutamic acid (VGSE)tetra-peptide comprised of valine, glycine, serine, and glutamic acid (VGSE)

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ES mass spectrum for VGSE

The base beak is the [M + 1]+ ion at m/z 391 the sodium adduct [M + 23]+ is nearly 90% the sodium adduct, [M + 23]+, is nearly 90% of the base peak. In addition there is some useful In addition, there is some useful fragmentation information characteristic of each of the amino acids. For small peptides, it is not uncommon to find some helpful fragmentation, but for proteins it is less likelyproteins it is less likely.

473.2

An example of this type of sample analysis is h h / f h d

p yp p yshown in the m/z spectrum of the pentapeptide leucine enkephalin, YGGFL.

The molecular formula for this compound is The molecular formula for this compound is C28H37N5O7 and the calculated monoisotopic molecular weight is 555.2692 Da.

Th / h d i i / 556 1 The m/z spectrum shows dominant ions at m/z 556.1, which are consistent with the expected protonated molecular ions, (M+H)+, ( )Protonated molecular ions are expected because the sample was analysed under positive ionisation conditions conditions.

These m/z ions are singly charged, and so the m/z value is consistent with the molecular mass, as the value of z (number of charges) equals 1 value of z (number of charges) equals 1.

Hence the measured molecular weight is deduced to be 555.1 Da, in good agreement with the theoretical

l48

g gvalue.

Positive ESI-MS m/z spectrum of Positive ESI MS m/z spectrum of leucine enkaphalin, YGGFL

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The m/z spectrum also shows other ions of lower i t it ( 25 % f th / 556 1 i ) t / intensity (ca.. 25 % of the m/z 556.1 ions) at m/z 557.2.

These represent the molecule in which one 12C atom has l d 13 h

pbeen replaced by a 13C atom, because carbon has a naturally occurring isotope one atomic mass unit (Da) higher.

Th int nsit f th s is t pi i ns l t s t th The intensity of these isotopic ions relates to the relative abundance of the naturally occurring isotope multiplied by the total number of carbon atoms in the molecule. molecule.

Additionally the fact that the 13C ions are one Da higher on the m/z scale than the 12C ions is an

d h 1 d h h l g

indication that z = 1, and hence the sample ions are singly charged.

If the sample ions had been doubly charged then If the sample ions had been doubly charged, then the m/z values would only differ by 0.5 Da as z, the number of charges, would then be equal to 2.

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The m/z spectrum also contains ions at m/z 578.1, p ,some 23 Da higher than the expected molecular mass.

These can be identified as the sodium adduct ions, These can be dent f ed as the sod um adduct ons, (M+Na)+, and are quite common in electrospray ionisation.

Instead of the sample molecules being ionised by the addition of a proton H+ some molecules have the addition of a proton H , some molecules have been ionised by the addition of a sodium cation Na+.

Other common adduct ions include Other common adduct ions include K+ (+39) and NH4

+ (+18) in positive ionisation mode andCl- (+35) in negative ionisation mode.

Electrospray ionisation is known as a "soft" ionisation method as the sample is ionised by the m mp yaddition or removal of a proton, with very little extra energy remaining to cause fragmentation of the sample ions

51

the sample ons

In ESI, samples (M) with molecular masses up to ca. 1200 Da give rise to singly charged molecular-D g g y g mrelated ions,

usually protonated molecular ions of the formula (M+H)+ in positive ionisation mode, and p ,deprotonated molecular ions of the formula (M-H)- in negative ionisation mode.

(e.g. YGGFL)( g )

Samples (M) with molecular weights greater than ca. 1200 Da give rise to multiply charged molecular1200 Da give rise to multiply charged molecular-related ions such as

(M+nH)n+ in positive ionisation mode and (M H) i i i i i d(M-nH)n- in negative ionisation mode.

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Proteins have many suitable sites for protonation as all of the backbone amide nitrogen atoms could be all of the backbone amide nitrogen atoms could be protonated theoretically, as well as certain amino acid side chains such as lysine and arginine which contain primary amine functionalities contain primary amine functionalities.

An example of multiple charging, which is practically unique to electrospray ionisation, is presented in the

iti i i ti / t f th t i h positive ionisation m/z spectrum of the protein hen egg white lysozyme

53

Positive ESI-MS m/z spectrum of the Positive ESI-MS m/z spectrum of the protien hen egg white lysozyme

54

The sample was analysed in a solution of 1:1 (v/v) t it il 0 1% s f i id d th / acetonitrile : 0.1% aqueous formic acid and the m/z

spectrum shows a Gaussian-type distribution of multiply charged ions ranging from m/z 1101.5 to 2044 62044.6.

Each peak represents the intact protein molecule carrying a different number of charges (protons). y g ff um f g (p )The peak width is greater than that of the singly charged ions seen in the leucine enkephalin spectrum as the isotopes associated with these spectrum, as the isotopes associated with these multiply charged ions are not clearly resolved as they were in the case of the singly charged ions.

Th i di id l k i th lti l h d iThe individual peaks in the multiply charged seriesbecome closer together at lower m/z values and, because the molecular weight is the same for all of th k th ith h t l the peaks, those with more charges appear at lower m/z values than do those with fewer charges

55

The m/z values can be expressed as f ll sfollows:

m/z = (MW + nH+)/nwhere m/z = the mass-to-charge ratio marked on the abscissa of the spectrum;MW = the molecular mass of the samplen = the integer number of charges on the ionsH = the mass of a proton = 1.008 Da.

56

If the number of charges on an ion is known, then it is simply a matter of reading the m/z value from is simply a matter of reading the m/z value from the spectrum and solving the above equation to determine the molecular weight of the sample.

Usually the number of charges is not known, but can be calculated if the assumption is made that

any two adjacent members in the series of multiply any two adjacent members in the series of multiply charged ions differ by one charge.

For example, pif the ions appearing at m/z 1431.6 in the lysozyme spectrum have "n" charges, then the ions at m/z 1301 4 will have "n+1" charges then the ions at m/z 1301.4 will have n 1 charges, and the above equation can be written again for these two ions:

1431 6 (MW nH+)/n 1431.6 = (MW + nH+)/n and 1301.4 = [MW + (n+1)H+]/(n+1)

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Positive ESI-MS m/z spectrum of the Positive ESI-MS m/z spectrum of the protien hen egg white lysozyme

58

1431.6 = (MW + nH+)/n 1301.4 = [MW + (n+1)H+]/(n+1)

These simultaneous equations can be rearranged to exclude the MW term:

n(1431 6) nH+ = (n+1)1301 4 (n+1)H+n(1431.6) – nH+ = (n+1)1301.4 – (n+1)H+

and so: n(1431.6) = n(1301.4) +1301.4 – H+n(1431.6) n(1301.4) 1301.4 H

therefore: n(1431.6 – 1301.4) = 1301.4 – H+

and soand so:

n = (1301.4 – H+)/(1431.6 – 1301.4)n = 1300.4/130.2 = 10. n 1300.4/130.2 10.

Putting the value of n back into the equation:

1431.6 = (MW + nH+)/n( )gives 1431.6 x 10 = MW + (10 x 1.008) and so MW = 14,316 - 10.08therefore MW = 14 305 9 Da

59

therefore MW = 14,305.9 Da

The observed molecular mass is in good agreement with the theoretical molecular mass of hen egg l (b d t i ) f lysozyme (based on average atomic masses) of 14305.14 Da.

The individual isotopes cannot be resolved when the pions have a large number of charges, and so for proteins the average mass is measured.

This may seem long-winded but fortunately the This may seem long-winded but fortunately the molecular mass of the sample can be calculatedautomatically, or at least semi-automatically, by the processing software associated with the mass processing software associated with the mass spectrometer.

This is of great help for multi-component mixture l i h th / t ll t i analysis where the m/z spectrum may well contain

several overlapping series of multiply charged ions, with each component exhibiting completely different h t t

p g p ycharge states.

Using electrospray or nanospray ionisation, a mass accuracy of within 0.01% of the molecular mass

60

accuracy of within 0.01% of the molecular mass should be achievable, which in this case represents ± 1.4 Da.

The m/z spectrum of lysozyme has been convertedto a molecular mass profile using Maximum Entropy m m p f g m m E pyprocessing and the data are shown. (next slide)

The mass profile is dominated by a component of The mass profile is dominated by a component of molecular mass 14,305.7 Da, with a series of minor peaks at higher mass, which is usually indicative of s lt dd tisalt adducting e.g.

Na (M+23), K (M+39), H2SO4 or H3PO4 (M+98).

The molecular masses can be read easily and The molecular masses can be read easily and unambiguously, and a good idea of the purity of the protein is obtained on inspection of the molecular mass profile mass profile.

61

Molecular mass profile of lysozyme obtained by maximum entropy processing of the m z by maximum entropy processing of the m/z spectrum

62

Characteristic charge distribution of Characteristic charge distribution of native or denaturated protein

63

Proteins in their native state, or at least containing a i ifi t t f f ldi significant amount of folding,

tend to produce multiply charged ions covering a smaller range of charge states (say two or three).

These charge states tend to have fewer charges than an unfolded protein would have, due to the inaccessibility of many of the protonation sites.

In such cases, increasing the sampling cone voltage may provide sufficient energy for the protein to b f ld d d h

y p gy pbegin to unfold and create a wider charge state distribution centering on more highly charged ions in the lower m/z region of the spectrum. g p

The differences in m/z spectra due to the folded state of the protein are illustrated with the m/z state of the protein are illustrated with the m/z spectra of the protein apo-pseudoazurin acquired under different solvent conditions.

64

Analysis of the protein in 1:1 acetonitrile : 0.1% aqueous formic acid at pH2 gave a Gaussian-type distribution

ith lti l h d t t i f with multiply charged states ranging from n = 9 at m/z 1487.8 to n = 19 at m/z 705.3, centering on n = 15 (lower trace).

The molecular mass for this protein was 13,381 Da.

Analysis of the protein in water gave fewer charge Analysis of the protein in water gave fewer charge states, from

n = 7 at m/z 1935.8 (1921.7) to 11 / 1223 7 n = 11 at m/z 1223.7,

centering at n = 9 (upper trace).

Not only has the charge state distribution changed Not only has the charge state distribution changed, the molecular weight is now 13,444 Da which represents an increase of 63 Da and indicates that copper is remaining bound to the protein copper is remaining bound to the protein.

Many types of protein complexes can be observed in this way, including protein-ligand, protein-peptide,

t i t l d t i RNA l l65

y p p p pprotein-metal and protein-RNA macromolecules

Water: fewer charge state

MW= 13,444

n = 7

Positive ESI-MS m/z spectra of

MW=13,381

Positive ESI MS m/z spectra of the protein apo-pseudoazurin

66

n = 9

Mass Analyzers

Magnetic Sector Mass S t tSpectrometersQuadrupole Mass Spectrometersp pIon Trap Mass SpectronletersTime of Flight Mass SpectrometerTime-of-Flight Mass SpectrometerFourier Transform Mass S t tSpectrometerTandem Mass Spectrometry

67

p y

Mass Analyzers

separates the mixture of ions that are generated during the ionization step by generated during the ionization step by m/z in order to obtain a spectrum

68

Summary of Mass Analyzers.

69

Magnetic Sector gMass Spectrometers

magnetic sector mass spectrometer (MS-MS) uses a magnetic field to deflect moving ions g garound a curved path

Separation of ions occurs based on the mass/charge ratio with lighter ions deflected to mass/charge ratio with lighter ions deflected to a greater extent than are the heavier ions.Resolution depends on each ion entering the ma netic field (fr m the s urce) with the same magnetic field (from the source) with the same kinetic energy, accomplished by accelerating the ions (which have a charge z) with a voltage VE k E Each ion acquires kinetic energy E

E = zV = mv2/2

70v is the velocity of the ion


When an accelerated ion enters the magnctic field (B). it experiences a deflecting force ( ) p g(Bzv), which bends the path of the ion orthogonal to its original direction. Th i i li i i l f The ion is now traveling in a circular part of radius r, given by

r = mv/ Bzr mv/ BzThe two equations can be combinedto give the familiar magnetic sector equation:

/ B2 2/2Vm/z = B2r2/2V

E = zV = mv2/2

71r is the radius of curvature of the pat


the greater the value of mlz, the l h di f h d hlarger the radius of the curved pathBecause the radius of the Because the radius of the instrument is fixed, the magnetic field is scanned to bring the ions field is scanned to bring the ions sequentially into focus.

72

73

Ouadrupole pMass Spectrometers

consists of four cylindrical (or of hyperbolic cross-section) rods (100-200hyperbolic cross section) rods (100 200mm long) mounted parallel to each other,at the corners of a square.A constant DC voltage modified by a radio frequency voltage is applied to the rods. q y g ppIons are introduced to the "tunnel" formed by the four rods of the formed by the four rods of the quadrupole in the center of the square at one end to the rods, and travel down the axis

74axis.

Ouadrupole pMass Spectrometers

For any given combination of DC voltage and modified voltage applied at the and modified voltage applied at the appropriate frequency, only ions with a certain mlz value possess a stablet j t d th f bl t trajectory and therefore are able to pass all the way to the end of the quadrupole to the detector to the detector.

All ions with different m/z values travel unstable or erratic paths and collide with pone of the rods or pass outside the quadrupole.

75

Schematic representation of a quadrupole Schematic representation of a quadrupole "mass filter" or ion separator.

76

Ion Trap Mass Spectrometer

EI spectra obtained with an ion trap f ll h bl ith are now fully searchable with

commercial databases.more sensitive than the quadrupoleroutinely configured to carry out y g ytandem experiments“trap” ions for relatively long trap ions for relatively long periods of time,

77


The ion trap generally consists of three electrodes,electrodes,

one ring electrode with a hyperbolic inner surfacetwo hyperbolic endcap electrodes at either end (across section of an ion trap is found) cross section of an ion trap is found)

The ring electrode is operated with a sinusoidal radio frequency field sinusoidal radio frequency field The endcap may be operated

t d t ti l at ground potential, or with either a DC or an AC voltage.

78


by controlling the three parameters of

RF voltage RF voltage, AC voltage, DC voltage,

a wide variety of experiments can be a wide variety of experiments can be run quite easily

79

Cross sectional view of an ion trap.

80


81

There are three basic modes in which the ion trapFirst, when the ion trap is operated with

a fixed RF voltage and DC bi b t th d d i l t dno DC bias between the endcap and ring electrodes,

all ions above a certain cutoff m/z ratio will be trapped.

As the RF voltage is raised, the cutoff m/z is increased in a controlled manner and the ions are sequentially ejected and detected. jThe result is the standard mass spectrum and this procedure is called the "mass-selective instability“ mode of operation of operation.

The maximum RF potential that can be applied between the electrodes limits the upper mass range in this mode the electrodes limits the upper mass range in this mode. Ions of mass contained beyond the upper limit are removed after the RF potential is brought back to zero.

82


The second mode of operation uses a DC potential across the endcaps; p

the general result is that there is now both a low and high-end cutoff (m/z) of ionsions.The possibilities of experiments in this mode ofoperation are tremendous, and most operations with the ion trap use this mode with the ion trap use this mode. As few as one ion mass can be selected. Selective ion monitoring is an important use of this

d f ti p

mode of operation. There is no practical limit on the number of ions masses that can be selected.

83

Ion Trap Mass SpectrometerThe third mode of operation is similar to the second, with the addition of an auxiliary oscillatory field between the endcap electrodes field between the endcap electrodes, result in adding kinetic energy selectively to a particular ion. pWith a small amplitude auxiliary field, selected ions gain kinetic energy slowly, during which time they usually undergo a fragmenting collision; the result usually undergo a fragmenting collision; the result can be a nearly 100% MS-MS efficiency. If the inherent sensitivity of the ion trap is considered along with the nearly 100% tandem considered along with the nearly 100% tandem efficiency, the use of the ion trap for tandem MSexperiment greatly outshines the so called ”triple quad”quad

84

Time-of-Flight (TOF)g ( )Mass Spectrometer

Ions are accelerated through a potential (V) and are then allowed to ”drift” down a tube to a detector. If the assumption is made-that

all of the ions arriving at the beginning of the drift all of the ions arriving at the beginning of the drift tube have the same energy given by zeV = mv2/2,

then ions of different mass will have different velocities: v = (2zeV/m)1/2velocities: v (2zeV/m)

If a spectrometer possesses a drift tube of length L, the time of flight for an ion is given by:

t (L2 /2 V)1/2 t = (L2m/2zeV)1/2

from which the mass for a given ion can be easily calculated.

85


Time-of-flight (TOF) is the least complex l i t f it thmass analyzer in terms of its theory

Ions are given a defined kinetic energyand allowed to drift through a field-free region (0.5 to several meters)The time ions arrive at the detector is measured and related to the m/z ratio

86


The critical aspect of this otherwise i l i t t i th d t d simple instrument is the need to produce

the ions at an accurately known start time nd p siti nand position.

These constraints generally limit TOF l d spectrometers to use pulsed ionization

techniques, which include plasma and laser desorption (e.g., MALDI, matrix assisted laser desorption ionization).

87


The resolution of TOF instruments is usually less than 20,000 because some variation in ion energy , gyis unavoidable. Also, since the difference in arrival times at the detector can bc less than 10-7 s fast electronics detector can bc less than 10 s, fast electronics are necessary for adequate resolution. On the positive side, the mass range of thcse instruments is unlimited and like quadrup lesinstruments is unlimited, and, like quadrupoles,they have excellent sensitivity due to lack of resolving slits.

l lThus, the technique is most useful for large biomolecules.

88

89

Fourier TransformMass Spectrometer

ions are held in a cell with an electric trapping potential within a strong magnetic field. p g gWithin the cell, each ion orbits in a direction perpendicular to the magnetic field, with a frequency proportional to the ion’s m/z.f qu n y p p n n m/zA radiofrequency pulse applied to the cell brings all of the cycloidal frequencies into resonancesimultaneously to yield an interferogramsimultaneously to yield an interferogram,conceptually similar to the free induction decay (FID) signal in NMR or the interferogram generated in FTIR experiments in FTIR experiments. The interferogram, which is a time domain spectrum,is Fourier transformed into a frequency domainspectrum which then yields the conventional mlz

90

spectrum, which then yields the conventional mlzspectrum.


Because it is operated at fixed magnetic field strength extremely high field field strength, extremely high field superconducting magnets can be used. Also, because mass range is directly , g yproportional to magnetic field strength, very high mass detection is possible.

Fi ll i ll f th i f i l Finally, since all of the ions from a single ionization event can be trapped and analyzed the method is very sensitive and analyzed, the method is very sensitive and works well with pulsed ionization methods.

91


The most compelling aspect of the method is its high resolution making method is its high resolution. making FT mass spectrometers an attractive alternative to other mass analyzers. alternative to other mass analyzers. The FT mass spectrometer can be coupled to chromatographic coupled to chromatographic instrumentation and various ionization methods, which means that it can be

il d i h ll l leasily used with small molecules.

92

Tandem Mass Spectrometry p y(MS-MS)

MS-MS (“MS squared”) is useful in studies with both known and unknown compounds; with both known and unknown compounds; with certain ion traps, MS to the nth (MS(n))is possible where n = 2 to 9.

In practice, n rarely exceeds 2 or 3.

With MS-MS, a "parent“ ion from the initial f t ti (th i iti l f t ti fragmentation (the initial fragmentation gives rise to the conventional mass spectrum) is selected and allowed or induced spectrum) is selected and allowed or induced to fragment further thus giving rise to ”daughter" ions.

93


In complex mixtures, these daughter ions provide unequivocal evidence for the presence of a known compound. p pFor unknown or new compounds, these daughter ions provide potential for daughter ions provide potential for further structural information.

94


One popular use of MS-MS involves ionizing a crude sample, selectively ”fishing out” an ion u mp , y f g ucharacteristic for the compound under study, and obtaining the diagnostic spectrum of the daughter ions produced from that ion daughter ions produced from that ion. In this way, a compound can be unequivocally detected in a crude sample, with no prior h m t phi ( th s p ti n st ps)chromatographic (or other separation steps)

being required.enables specific compounds to be detected in enables specific compounds to be detected in complex mixtures on account of their specific and characteristic fragmentation patterns. Thus MS MS can be a very powerful screening

95Thus, MS-MS can be a very powerful screening tool.


This type of analysis alleviates the need for complex separations of mixtures for for complex separations of mixtures for many routine analyses.

For instance, the analysis of urine samplesf h ( f th i l h

y pfrom humans (or from other animals such as race horses) for the presence of drugs or drug metabolites can be carried out routinely

h l ( f g y

on whole urine (i.e., no purification or separation) by MS-MS.

For unknown compounds these daughter For unknown compounds, these daughter ions can provide structural information as well.

96


One way to carry out MS-MS is to link t l i i t two or more mass analyzers in series to produce an instrument capable of

l ti i l i d i i h selecting a single ion, and examining how that ion (either a parent or daughter ion) fr m ntsfragments.For instance, three quadrupoles can be linked (a so called "triple quad") to produce a tandem mass spectrometer.

97

Stage q0: focusing quadrupole; Q1, Q3: mass analyzing quadrupoles; q2: collision cell.

A triple quadrupole instrument mbi ti f t m ss d l m ss filt s a combination of two mass quadrupole mass filters

(tandem mass spectrometry) separated by a collision cell which is also a quadrupole operating collision cell which is also a quadrupole operating in RF-only mode 98


A frequently used instrument of this type uses quadrupoles as analysersuses quadrupoles as analysers.

In this arrangement, th fi st d p l s l ts sp ifi i n f the first quadrupole selects a specific ion for further analysis, the second quadrupole functions as a collision the second quadrupole functions as a collision cell (collision induced decomposition, CID) and is operated with radiofrequency only,

lthe third quadrupole separates the product ions, to produce a spectrum of daughter ions.

99

Settings of the Q1 and Q3 quadrupoles for the various Settings of the Q1 and Q3 quadrupoles for the various scan modes of a triple quadrupole mass spectrometer

A product ion scan can obtain structural information of a i i

100

pgiven precursor ion A precursor ion scan is more suited to find structural homologues in a complex mixture.

Bosentan is an oral duel endothelin receptor antagonist approved for the use in arterial antagonist approved for the use in arterial hypertension.

Bosentan (M 551) has two metabolites corresponding to the tert-butyl hydroxylation product (M 567) and the dealkylation of the methoxy group to form the the dealkylat on of the methoxy group to form the phenol (M 537).

S l ti f th f t t / 280 fish t Selection of the fragment at m/z 280 can fish out precursor ions corresponding only to bosentan and these two metabolites

A similar result is obtained with the constant-neutral loss scan mode which is based on neutral loss neutral loss scan mode which is based on neutral loss of 44 units.

101

102

A: Q1 full-scan spectrum of bosentan M+H+ m/z 552bosentan, M+H , m/z 552,

its demethylated metabolite, M+H+, m/z 538and its hydroxylated metabolite M+H+, m/z 568

B: product ion spectrum of bosentan,C i tC: precursor ion spectrumD: neutral loss spectrum. El t i i ti i i iti i Electrospray ionization is in positive ion

mode.

103


In order for an instrument to carry out MS-MS, it must be able to do the three operations outlined b

pabove. As we have seen however, ion-trap systems capable of MS-MS and MS(n) do not use a tandem of MS MS and MS do not use a tandem arrangement of mass analyzers at all, but rather use a single ion trap for all three operations simultaneously. yAs has already been stated, these ion-trap tandem mass spectrometer experiments are very sensitive and are now user friendlyand are now user friendly.The ion trap brings the capability for carrying out MS-MS experiments to the benchtop at relatively low cost

104

low cost.


105

Principle of MS/MS: an ion M1 isselected by the first spectrometerMS1 fragmented through collisionMS1, fragmented through collision,and the fragments are analysed bythe second spectrometer, MS2. Thus ions with a selected m/z value Thus ions with a selected m/z value, observed in a standard source spectrum,can be chosen and fragmented

t bt i th i d t iso as to obtain their product ionspectrum

106

magnetic sector is labelled B andelectric sector is labelled E.Electromagnetic AnalysersElectromagnetic Analysers

Structure elucidation by MS/MS of a nodulation factor. L ft: m/ lu s bs d b FAB f m ch m t phic f cti n fLeft: m/z values observed by FAB from a chromatographic fraction ofnodulation factors. Right: the m/z 1244 ion is selected as precursor and fragmented by B/E linked scan in a magnetic instrument

107

linked scan in a magnetic instrument. The clear-cut fragmentation at the glycosylic bonds allows straightforward sequence assignment as shown by the fragmentation scheme.

The two analysers are separated by a collision cell into which an inert gas (e.g. argon, xenon) is admitted to

llid ith th l t d l i d b i b t collide with the selected sample ions and bring about their fragmentation.

The analysers can be of the same or of different types, the most common combinations being:

quadrupole quadrupolequadrupole - quadrupole

magnetic sector - quadrupole

magnetic sector - magnetic sector

d l i f fli hquadrupole – time-of-flight.

Fragmentation experiments can also be performed on Fragmentation experiments can also be performed on certain single analyser mass spectrometers such as ion trap and time-of-flight instruments, the latter type using a post-source decay experiment to effect the using a post source decay experiment to effect the fragmentation of sample ions

108

Tandem mass spectrometry Tandem mass spectrometry analyses

The basic modes of data acquisition for t d t t i t tandem mass spectrometry experiments are as follows:

Product or daughter ion scanningPrecursor or parent ion scanning

l l Constant neutral loss scanningSelected/multiple reaction monitoring

109

Product or daughter ion scanningthe first analyser is used to select user-specified sample ions arising from a particular component;

usually the molecular-related (i.e. (M+H)+ or (M-H)-) ions.usually the molecular related (i.e. (M H or (M H) ) ions.

These chosen ions pass into the collision cell, are bombarded by the gas molecules which cause fragment ions to be formed to be formed, and these fragment ions are analysed i.e. separated according to their mass to charge ratios, by the second analyser.

All the fragment ions arise directly from the precursor All the fragment ions arise directly from the precursor ions specified in the experiment, and thus produce a fingerprint pattern specific to the compound under investigation investigation.

This type of experiment is particularly useful for providing structural information concerning small

l l d f d 110

p g gorganic molecules and for generating peptide sequenceinformation

Precursor or parent ion scanning

the first analyser allows the transmission of all sample ions, p ,the second analyser is set to monitor specific fragment ions, which are generated by bombardment of the sample ions with the collision bombardment of the sample ions with the collision gas in the collision cell. This type of experiment is particularly useful for m nit rin r ups f c mp unds c ntained within a monitoring groups of compounds contained within a mixture which fragment to produce common fragment ions, e.g.

glycosylated peptides in a tryptic digest mixture,aliphatic hydrocarbons in an oil sample, or glucuronide conjugates in urine.

111

g j g

Constant neutral loss scanning

this involves both analysers scanning, or collecting data, across the whole m/z range, but the two are off-set so g ,that the second analyser allows only those ions which differ by a certain number of mass units (equivalent to a neutral fragment) from the ions transmitted through f g ) f gthe first analyser. e.g. This type of experiment could be used to monitor all of the carboxylic acids in a mixture.

Carboxylic acids tend to fragment by losing a (neutral) molecule Carboxylic acids tend to fragment by losing a (neutral) molecule of carbon dioxide, CO2, which is equivalent to a loss of 44 Da or atomic mass units.

All ions pass through the first analyser into the collision cell. p g yThe ions detected from the collision cell are those from which 44 Da have been lost.

112

Selected/multiple reaction monitoring

both of the analysers are static in this case as user-selected specific ions are transmitted through p gthe first analyser and user-selected specific fragments arising from these ions are measured by the second analyser. yThe compound under scrutiny must be known and have been well-characterised previously before this type of experiment is undertaken type of experiment is undertaken.

This methodology is used to confirm unambiguously the presence of a compound in a matrix e.g. drug t ti ith bl d i ltesting with blood or urine samples.

It is not only a highly specific method but also has very high sensitivity.

113

y g y

Analysis of Biomoleculesy

Peptide sequencingPept de sequenc ngMetabolomics

Peptide Sequencing by Tandem p q g yMass Spectrometry

The most common usage of MS-MS in biochemical areas is the product or daughter ion scanningg gexperiment which is particularly successful for peptide and successful for peptide and nucleotide sequencing

115

Peptide sequencing: p q gH2N-CH(R’)-CO-NH-CH(R”)-CO2H

There are three different types of bonds that can fragment along the amino acid backbone: g g

the NH-CH, CH-CO, and CO-NH bonds CO NH bonds.

Each bond breakage gives rise to two species, one neutral and th th h d d l th h d i i the other one charged, and only the charged species is monitored by the mass spectrometer.

The charge can stay on either of the two fragments d di th h i t d l ti t depending on the chemistry and relative proton affinity of the two species.

116


Hence there are six possible fragment ions for each amino acid residue and these are labelled as in the diagram, with

the a, b, and c" ions having the charge retained on the N-terminal fragment, and g ,the x, y", and z ions having the charge retained on the C-terminal fragment.

The most common cleavage sites are at the CO-The most common cleavage sites are at the CONH bonds which give rise to the b and/or the y” ions.

The mass difference between two adjacent b The mass difference between two adjacent b ions, or y”; ions, is indicative of a particular amino acid residue (see Table of amino acid residues)

117residues).

Table of amino acid residuesSymbol Structure Mass (Da)

Table of amino acid residues

Ala A -NH.CH.(CH3).CO- 71.0Arg R -NH.CH.[(CH2)3.NH.C(NH).NH2].CO- 156.1Asn N -NH.CH.(CH2CONH2).CO- 114.0Asp D -NH.CH.(CH2COOH).CO- 115.0Cys C -NH.CH.(CH2SH).CO- 103.0Gln Q -NH.CH.(CH2CH2CONH2).CO- 128.12 2 2

Glu E -NH.CH.(CH2CH2COOH).CO- 129.0Gly G -NH.CH2.CO- 57.0y 2

His H -NH.CH.(CH2C3H3N2).CO- 137.1Ile I -NH.CH.[CH.(CH3)CH2.CH3].CO- 113.1

118

[ ( 3) 2 3]

Table of amino acid residuesSymbol Structure Mass (Da)Leu -NH.CH.[CH2CH(CH3)2].CO- 113.1[ 2 ( 3)2]Lys K -NH.CH.[(CH2)4NH2].CO- 128.1Met M -NH.CH.[(CH2)2.SCH3].CO- 131.0[( 2)2 3] .Phe F -NH.CH.(CH2Ph).CO- 147.1Pro P -NH.(CH2)3.CH.CO- 97.1Pro P NH.(CH2)3.CH.CO 97.1Ser S -NH.CH.(CH2OH).CO- 87.0Thr T -NH CH [CH(OH)CH3) CO- 101 0Thr T NH.CH.[CH(OH)CH3).CO 101.0Trp W -NH.CH.[CH2.C8H6N].CO- 186.1Tyr Y -NH CH [(CH ) C H OH] CO- 163 1

119

Tyr Y -NH.CH.[(CH2).C6H4.OH].CO- 163.1Val V -NH.CH.[CH(CH3)2].CO- 99.1

Imm i m d l t d i m ss s ft F li k Immonium and related ion masses after Falick, 1993 and Papayannopoulos, 1995.

Residue 3-letter code 1-letter Immonium ion Related ionResidue 3 letter code 1 letter Immonium ion Related ionAlanine Ala A 44Arginine Arg R 129 59,70,73,8Asparagin Asn N 87 70Aspartic Asp D 88 70Aspartic Asp D 88 70Cysteine Cys C 76Glutamic Glu E 102Glutamine Gln Q 101 56,84,129Glycine Gly G 30Glycine Gly G 30Histidine His H 110 82,121,123,Isoleucine Ile I 86 44,72Leucine Leu L 86 44,72

4Lysine Lys K 101 70,84,112,1Methionin Met M 104 61Phenylalan Phe F 120 91Proline Pro P 70Serine Ser S 60Threonine Thr T 74Tryptopha Trp W 159 77,117,130,Tyrosine Tyr Y 136 91,107

120* Bold face indicates strong signals, italic indicates weak.

y yValine Val V 72 41,55,69

Peptide sequencing by tandem mass spectrometry - backbone cleavages

A, B, and C ions:N-terminal ffragment

x, y", and z ions:C-terminal fragment

121


The extent of side-chain fragmentationdetected depends on the type of detected depends on the type of analysers used in the mass spectrometer.

A magnetic sector-magnetic sectorg ginstrument will give rise to high energycollisions resulting in many different types of side chain cleavages types of side-chain cleavages.

Quadrupole-quadrupole and quadrupole –time-of-flight mass spectrometers time of flight mass spectrometers generate low energy fragmentations with fewer types of side-chain fragmentations.

122

Immonium ions

labelled "i" appear in the very low m/z range of the MS-MS spectrum.

l f h l d h f d b an internal fragment with just a single side chain formed by a combination of a type and y type cleavage is called an immoniumion.Each amino acid residue leads to a diagnostic immonium ion with Each amino acid residue leads to a diagnostic immonium ion, with the exception of

the two pairs leucine (L) and iso-leucine (I), and lysine (K) and glutamine (Q) lysine (K) and glutamine (Q),

which produce immonium ions with the same m/z ratio, i.e. m/z 86 for I and L, m/z 101 for K and Q.

The immonium ions are useful for detecting and confirming many he mmon um ons are useful for detect ng and conf rm ng many of the amino acid residues in a peptide,

although no information regarding the position of these amino acid residues in the peptide sequence can be ascertained from the immonium ions

123

immonium ions.

example of an MS/MS daughter or example of an MS/MS daughter or product ion spectrum

The molecular mass of the peptide was measured using standard mass spectrometric techniques and found to be 680.4 Da, ,the dominant ions in the MS spectrum being the protonated molecular ions (M+H)+ at m/z 681.4.

These ions were selected for transmission through the first These ions were selected for transmission through the first analyser, then fragmented in the collision cell and their fragments analysed by the second analyser to produce the following MS/MS spectrum. following MS/MS spectrum.

The sequence (amino acid backbone) ions have been identified, and in this example the peptide fragmented predominantly at the CO-NH bonds and gave both b and y" predominantly at the CO NH bonds and gave both b and y ions.

Often either the b series or the y" series predominates, sometimes to the exclusion of the other

124

m m f

The b series ions have been labelled with blue vertical linesh h l ll d h d l lthe y" series ions have been labelled with red vertical lines.

The mass difference between adjacent members of a The mass difference between adjacent members of a series can be calculated e.g.

b3-b2 = 391.21 - 262.16 = 129.05 Da hi h i i l t t l t i (E) i id id which is equivalent to a glutamine (E) amino acid residue;

and similarly y4 - y3 = 567.37 - 420.27 = 147.10 Da which is equivalent to a phenylalanine (F) residue. q p y ( )

In this way, using either the b series or the y” series, th i id f th tid b the amino acid sequence of the peptide can be determined and was found to be NFESGK the y” series reads from right to left!The immonium ions at m/z 102 merely confirm the presence of the glutamine (E) residue in the peptide.

125

B2B3

B2

Y4

Y3

126

A protein identification studyp ywould proceed as follows

a. The protein under investigation would be analysed by mass spectrometry to generate a molecular mass to within an accuracy of 0.01%.

b. The protein would then be digested with a suitable enzyme. Trypsin is useful for mass spectrometric studies because Trypsin is useful for mass spectrometric studies because each proteolytic fragment contains a basic arginine (R) or lysine (K) amino acid residue, and thus is eminently suitable for positive ionisation mass spectrometric analysis. p p y

The digest mixture is analysed - without prior separationor clean-up by mass spectrometry to produce a rather or clean up by mass spectrometry to produce a rather complex spectrum from which the molecular weights of all of the proteolytic fragments can be read.

127

protein identification study

This spectrum, with its molecular weight information, is called a peptide map. p p p

If the protein already exists on a database, then the peptide map is often sufficient to confirm the protein

For these experiments the mass spectrometer would be operated in the ”MS” mode, whereby the sample is sprayed and ionised from the nanospray needle and the p y f p yions pass through the sampling cone, skimmer lenses,Rf hexapole focusing system, and the first (quadrupole) analyser. (qu up ) y .

The quadrupole in this instance is not used as an analyser, merely as a lens to focus the ion beam into the second (time-of-flight) analyser which separates the ions

128

(time-of-flight) analyser which separates the ions according to their m/z ratio.

protein identification studyc. With the digest mixture still spraying into the mass spectrometer,

the Q-Tof mass spectrometer is switched into “MS/MS” mode.

The protonated molecular ions of each of the digest fragments The protonated molecular ions of each of the digest fragments can be independently selected and transmitted through the quadrupole analyser, which is now used as an analyser to transmit solely the ions of interest into the collision cell which lies i b t th fi t d d linbetween the first and second analysers.

An inert gas such as argon is introduced into the collision cell and the sample ions are bombarded by the collision gas moleculeswhich cause them to fragment which cause them to fragment.

The optimum collision cell conditions vary from peptide to peptide and must be optimised for each one.

The fragment (or daughter or product) ions are then analysed by The fragment (or daughter or product) ions are then analysed by the second (time-of-flight) analyser.

In this way an MS/MS spectrum is produced showing all the fragment ions that arise directly from the chosen parent or

129

fragment ions that arise directly from the chosen parent or precursor ions for a given peptide component.

Q TOF mass spectrometer operating in MS Q-TOF mass spectrometer operating in MS (upper) and MS/MS mode (lower) modes

130


An MS/MS daughter (or fragment, or product)

ion spectrum is produced for each of the ion spectrum is produced for each of the components identified in the proteolytic digest.

Varying amounts of sequence information can be gleaned from each fragmentation spectrum, and the spectra need to be interpreted carefully.

Some of the processing can be automated but in Some of the processing can be automated, but in general the processing and interpretation of spectra will take longer than the data acquisition f d l bl d b dp g

if accurate and reliable data are to be generated.

131


The amount of sequence information generated will vary from one peptide to generated will vary from one peptide to another

Some peptide sequences will be confirmed Some peptide sequences will be confirmed totally, other may produce a partial sequence of, say, y p p q , y,4 or 5 amino acid residues.

Often sequence "tag" of 4 or 5 residues is ff h d bsufficient to search a protein database

and confirm the identity of the protein.

132

protein study

HPLC represents an attractive alternative to two-dimensional electrophoresis for the two dimensional electrophoresis for the separation of both proteins and peptides because of its chromatographic resolving power,

d ibilit d its tibilit ith MS reproducibility and its compatibility with MS detection.

Th f l idi i l h h f The use of multidimensional chromatography for the separation of complex protein and peptide mixtures has consequently seen increased use in mixtures has consequently seen increased use in proteomics studies

133

134

protein study

Example of a LC-MS/MS analysis of a C. elegans extract extract.

(A) Fraction 2, 4 mM KCl salt elution on the strong cation exchange column.

(B) Full scan MS spectrum of the peak eluting at RT 26.3 min in (A).

(C) P d t i t f th d bl h d (C) Product ion spectrum of the doubly charged precursor of (B) at m/z 784.8.

With the help of bioinformatic tools the product With the help of bioinformatic tools the product ion spectrum can be automatically interpreted.

135

Peptide sequencing in summary

Peptides fragment along the amino acid backbone to give sequence information. g qPeptides ca. 2500 Da or less produce the most useful data.

The amount of sequence information varies from one The amount of sequence information varies from one peptide to another.

Some peptides can generate sufficient information for a full s qu nc t b d t rmin d; sequence to be determined; others may generate a partial sequence of 4 or 5 amino acids.

A t i di t b l d ti ti A protein digest can be analysed as an entire reaction mix, without any separation of the products, from which individual peptides are selected and analysed b th t t t t

136by the mass spectrometer to generate sequence information.

Peptide sequencing in summaryAbout 4 µL of solution is required for the analysis of the digest mixture, with a concentration based on the original protein of ca. 1-10 pmol/µL. g p p µMS/MS sequencing is a sensitive technique consuming little sample.

Sometimes the full protein sequence can be verified; Sometimes the full protein sequence can be verified; some proteins generate sufficient information to cover only part of the sequence. 70-80% coverage is r s n blreasonable.

Often a sequence "tag" of 4/5 amino acids from a single proteolytic peptide is sufficient to identify the p y p p yprotein from a database.

The final point in this summary means that mass spectrometers have been found to be extremely useful

137

spectrometers have been found to be extremely useful for proteomic studies, as illustrated below.


The proteomics procedure usually involves excising individual spots from a 2-D gel and independently p f m D g p yenzymatically digesting the protein(s) contained within each spot, before analysing the digest mixture by mass

t t i th tli d bspectrometer in the manner outlined above.

Electrospray ionisation or MALDI could be used at this stepstep.

The initial MS spectrum determining the molecular masses of all of the components in the digest mixture masses of all of the components in the digest mixture can often provide sufficient information to search a database using just several of the molecular weights g j gfrom this peptide map.

138


If the database search is not fruitful, either because because

the protein has not been catalogued, is previously uncharacterised, or h d h h the data are not accurate or comprehensive enough

to distinguish between several entries in the database, then further information is required., q

This can be achieved by sample clean-up and then MS/MS studies to determine the amino acid sequences of the individual proteolytic peptides contained in the digest mixture, with

139

p p g m u , wwhich further database searching can be carried out.

Metabolomics

140140

Metabolomics

Metabolomics is the ‘omics’ science of metabolism. This term has been defined in analogy with This term has been defined in analogy with genomics, transcriptomics and proteomics genomics

Genome: the entire collection of the genes of an Genome: the entire collection of the genes of an organism. Transcriptomics looks at the transcriptome that Transcriptomics looks at the transcriptome that is the complete set of mRNA transcripts Proteomics looks at the proteome that is the Proteomics looks at the proteome that is the expressed set of proteins that are encoded by the genome.

141141

MetabolomicsMetabolome can be defined as the complete set of small molecules (non-polymeric compounds with a molecular

i h l h b 1000 D ) h i l d i weight lower than about 1000 Da) that are involved in general metabolic reactions and that are biosynthesized by a cell, tissue or organisma cell, tissue or organismHigher mass compounds are often polymers and their systematic identifications are termed according to the class of compounds, such as the ‘glycome’ for example.Metabolomics is the study that

th id tifi ti d tifi ti f th covers the identification and quantification of the metabolome under a given set of conditions. It also studies the dynamic changes in the metabolomey g

142142

Metabolomics: applications

By accurately measuring the metabolome changes between between

healthy and diseased patients, d seased pat ents,

biomarker compounds of various diseases can be identified and become diagnostic for these diseases. gBy mapping these changes to known metabolic pathways,

enzymes that are responsible for these changes can be deduced and thus the enzyme that is critical to a disease can be identified.f .

143143


To validate this enzyme as a good candidate for drug discovery it can be inactivated by an drug discovery, it can be inactivated by an inhibitor or a genetic modification. The induced effect on the metabolome can be The induced effect on the metabolome can be compared with the disease. If the metabolomes are similar, then the If the metabolomes are similar, then the inactivated enzyme is a good candidate. Unfortunately, there are few detailed y,descriptions of complete metabolome analysis in the literature.

144144


Identify endogenous substrates of f tt l id h d lenzyme fatty acyl amide hydrolase

(FAAH) that regulates several brain lipidsth t h i t ti h l i l that have interesting pharmacological properties including effects on the

t l f icontrol of pain.by analysis of metabolomes from wild-

d dtype and enzyme-inactivated organisms

145145


Knockout mice lacking the gene to synthesize FAAH would accumulate all the possible FAAH would accumulate all the possible endogenous substrates of this enzyme. Thus a comparison of the metabolomes of Thus, a comparison of the metabolomes of normal and knockout mice would allow the identification of all substrates of FAAH, present pmainlAfter exhaustive extraction from these tissues, the mixture was first analysed by HPLC/ESI/MS in both the positive and negative ion mode.

146146


a comparison of the metabolomes of normal and knockout mice would allow

147

pthe identification of all substrates of FAAH, present mainly in the brain and spinal cord. 147

Ratio of the ion intensities FAAH(−/−)/FAAH(+/+) observed at the different masses during elution of observed at the different masses during elution of extracts from the brain and spinal cord of knockout mice (FAAH(−/−)) and normal mice (FAAH(+/+)). This plot reveals two regions where compounds are in much higher concentration in knockout mice. Th fi i d k l f The first region corresponds to a known class of substrates for FAAH that are related to ethanolamine amides (NAE) amides (NAE).

This class of substrates is much larger than previously described because several unrecognized compounds of this g pclass, such as the C24:1 compound, were also found. Very low-abundance compounds of this class, such as

d id (C20:4) ls d t t d anandamide (C20:4), were also detected. The second region is particularly interesting and corresponds to an unknown class of substrates for FAAHcorresponds to an unknown class of substrates for FAAH.

148148

AA

BB

149149

Identification and structural characterization of an unknown class of characterization of an unknown class of substrates for FAAH.

(A) By high-resolution mass spectrometrythe high accuracy mass measurements of a compound of this class gives an exact mass of 446 3310 446.3310 that corresponds to a molecular formula of C24H48NO4SC24H48NO4S.

(B) By MS/MS analysis, th st t f this mp nd is ssi n d s the structure of this compound is assigned as the C24:0 fatty acyl amide of taurine (NAT).

ONH2

150150HO

S

Otaurine

Drug MetabolismDuring drug discovery and drug development, it is important to establish how the body metabolizes a drugdrug

therefore rapid identification of metabolites from in vitro or in vivo samples becomes essential

l l lMetabolic stability of drugs is also an important parameter in drug discovery

hundreds of samples can be rapidly generated using in hundreds of samples can be rapidly generated using in vitro systems such as hepatocytes and microsomes. For structural elucidation, NMR is the technique of choice but it does not allow high throughput analysischoice, but it does not allow high throughput analysisand sensitivity is still in the microgram range. LC-MS has therefore become the technique of choice.

151

LC-MS data dependent analysis of vinpocetin in rat urineusing dynamic background substraction (DBS) on a triple quadrupole linear ion trap. (A) Full scan MS (survey scan) trace (A) Full scan MS (survey scan) trace.

(B) Enhanced product ion scan (dependent scan).

The major peak at 3.9 min corresponds to apovinpocetin,

the minor one at 2.9 min to the hydroxylation product of apovinpocetin (m/z 339)m m y y p f p p (m )

NN

O

O

inp tinvinpocetin

152

Schematic of online LC-MS analysis combined with fractioncollection into 96-well plate. pDepending on the online MS data, further MS experiments are performed with chip-based infusion at 200 nL min-1

153

Using predictive fragmentation Using predictive fragmentation software

The understanding of the fragmentation mechanism of the parent drug is very important for the of the parent drug is very important for the metabolite assignment.

The product ion spectrum of remikiren

Conventional spectra interpretation is time-consuming and the use of predictive fragmentation software such as Mass Frontier (High-Chem) can help to rationalize spectra ( g ) p pinterpretationIn the case of the fragment at m/z 282,

three different fragments are proposed by the software. three d fferent fragments are proposed by the software. Only accurate mass measurement with an accuracy better than 10 ppm allowed selection of the right fragment

154

(A) Product ion spectrum of remikiren obtained on a QqTOF(B) S ft di t d (B) Software-predicted fragments (Mass Frontier, HighChem) for theion at m/z 282ion at m/z 282

Advances in high resolution mass analyzers (TOF, FT-ICR, orbitrap) havegreatly improved the detection and greatly improved the detection and identification of metabolites based on accurate mass measurements. C33H50N4O6S: mass = 630.3451

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