Mass spectrometry basics

General scheme of Proteomic analysis

Protein Mixture

Peptide mixture Proteins

Peptides

M S Data

Identification

Digestion Separation

Separation Digestion

MS Analysis

Data Reduction Algorithm

Information from Mass spectrometryDetermination of Molecular weight.

Accuracy of 0.01% of the total molecular weight.

Changes in mass can be detected e.g. substitution of one amino acid for another, or a post-translational modification.

Structural information can be achieved by fragmenting the sample and analyzing the products generated.

Uses of mass spectrometerUsed in industry and academia for both routine and research purposes. Brief summary of the major mass spectrometric applications:

•Biotechnology: the analysis of proteins, peptides, oligonucleotides

•Pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism

•Clinical: neonatal screening, hemoglobin analysis, drug testing •Environmental: PAHs, PCBs, water quality, food contamination

•Geological: oil composition

Mass spectrometry for biochemists

Accurate molecular weight measurements:

confirmation of sample, determination of purity of a sample, verifying amino acid substitutions, detection of post-translational modifications, calculating number of disulphide bridges .

Reaction monitoring:

to monitor enzyme reactions, chemical modification, protein digestion

Amino acid sequencing:

sequence confirmation, de novo characterisation of peptides, identification of proteins by database searching with a sequence “tag” from a proteolytic fragment

Oligonucleotide sequencing:

the characterization or quality control of oligonucleotides

Protein structure:

protein folding monitored by H/D exchange, protein-ligand complex formation under physiological conditions, macromolecular structure determination

MS divided into 3 fundamental parts

Mass spectrometer

Data system

Ionization sourcee.g. electrospray(ESI),Matrix assisted laserDesorption(MALDI)

AnalyserMass to charge,m/ze.g. quadrupole,Time of flight, magnet, FT-ICR

Detectore.g. photomultiplier,Microchannel plate,Electron multiplier

Types of Mass spectrometry

Working of Mass Spectrometry

Divided into three fundamental parts:Ionization source Analyzer Detector

Sample is introduced in the ionization source where they are ionized. ( It is easier to manipulate ions than neutral molecules).

Ions separated according to their mass to charge ratio in the analyzer. The separated ions are detected and this signal sent to a data system where the m/z ratios are stored together with their relative abundance for presentation in the format of a m/z spectrum.

The separated ions are detected and this signal sent to a data system where the m/z ratios are stored together with their relative abundance for presentation in the format of a m/z spectrum.

Sample introduction

IonizationMinimize collisions, interferences

Separatemasses

Count ionsCollect results

Mass spectrometry Introduction

Sample introductionThe sample can be inserted directly into the ionization source, or can undergo some

type of chromatography en route to the ionisation source.

Gas source (lighter elements)dual inlet - sample purified and measured with standard gas at identical conditions

precisions ~ ±0.005%continuous flow - sample volatized and purified (by EA or GC) and injected into

mass spec in He carrier gas, standards measured before and after,precisions ~ 0.005-0.01%

Solid source (heavier elements)TIMS - sample loaded onto Re filament, heated to ~1500°C, precisions ~0.001%laser ablation - sample surface sealed under vacuum, then sputtered with laser

precisions ~0.01%

Inductively coupled plasma (all elements)ICPMS - sample converted to liquid form,

converted to fine aerosol in nebulizer,injected into ~5000K plasma torch

Matrix-Assisted Laser Desorption/Ionization (MALDI)

Used for nonvolatile & high Molecular analytes

biopolymers and oligomers

proteins, peptides, oligonucleotides, oligosaccharides

synthetic polymers

Inorganic, such a fullerenes

environmental compounds

kerogens, coal tars, humic acids, fulvic acids

MALDI Principle

A laser pulse is used for excitation

UV lasers cause electronic excitation

IR lasers cause vibrational excitation

Matrix molecules transfer energy

100-50,000 x [A]

low analyte fragmentation

matrices are selectable

TOF-MS is used for analysis

Matrices

Solid

co-crystallization of analyte and matrix

occasionally frozen solvents

Liquid

IR or UV absorbing liquid containing analyte

Two phase

liquids with absorbing solid

Common Matrices

α-cyno-4-hydroxycinnamic acid

2.5-dihydroxybenzoic acid Sinapinic acid

3-amino-4-hydroxybenzoic acid

Fragmentation

MALDI usually gives ‘molecular ions’

most often as protonated molecules

Control of fragmentation

differences in sublimation temperature of matrices control thermal excitation

PA and IP of a matrix affect energy transfer involved with protonation and electron transfer

collisions

extraction through neutral plume

residual gas

Ionization in MALDI

Ionization is separated into two divisions

primary ion formation

initial ions formed during laser pulse

frequently matrix molecules

secondary ion formation

ions formed during subsequent reactions

may be matrix-matrix reactions or matrix-analyte reactions

Resulting analyte ions are usually

protonated

Cationized

radical cations

Overview of Mass Spectrometry

Mass Spectrum

Mass Analyzer

Ionization M+/FragmentationSample Molecule (M)

Protonation : M + H+ MH Cationization : M + Cat+ MCat+

Deprotonation: MH M- + H+

Electron Ejection: M M+. + e-Electron Capture: M + e- M-.

Mechanism of Ionization

Matrix Assisted Laser Desorption Ionization (MALDI) (F. Hillenkamp, M. Karas, R. C. Beavis, B. T. Chait, Anal. Chem., 1991, 63, 1193), deals well with thermolabile, non-volatile organic compounds for the analysis of proteins, peptides, glycoprotein, oligosaccharides, and oligonucleotides.

Measures masses within 0.01% of the molecular weight of the sample, at least up to ca. 40,000 Da.

Based on the bombardment of sample molecules with a laser light to bring about ionisation.

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

Matrix transforms the laser energy into excitation energy for the sample, leads to sputtering of analyte and matrix ions from the surface of the mixture.

In this way energy transfer is efficient and also the analyte molecules are spared excessive direct energy that may otherwise cause decomposition.

Most commercially available MALDI mass spectrometers now have a pulsed nitrogen laser of wavelength 337 nm.

Matrix Assisted Laser Desorption Ionisation

"Somehow, a peak seems to have appeared." Tanaka reported at the weekly Monday team meeting on February 2nd, 1985, half a year after the project had started.

Six O'clock in the Evening on October 9th 2002 News arrived saying that Koichi Tanaka had won the Nobel Prize in Chemistry 2002 On October 9th, the Royal Swedish Academy of Sciences announced their decision to award the Nobel Prize in Chemistry 2002 to three people for their development of methods for identification and structure analyses of biological macromolecules. - Koichi Tanaka (at the time : Life Science Laboratory Assistant Manager of Shimadzu Corporation), Prof. John B. Fenn (Virginia Commonwealth University, USA) and Prof. Kurt Wuthrich (Swiss Federal Institute of Technology)

1. Sample is mixed in matrix and dried on target.

2.Target is introduced into high vacuum of MS.

3.Sample is irradiated with laser desorbing ions into the gas phase and the clock measuring the time of flight starts.

4.Ions are accelerated by an electric field to the same kinetic energy and they drift down the field free flight tube where they are separated in space

5.Ions strike the detector at different times depending on the mass to charge ratio of the ions

6.A data system controls all the parameters, acquires the signal vs. time and permits data processing

Schematic of a MALDI-TOF Experiment

Diagrammatic representation

Laser induced ionization

Sample probe

vacuum

optics

Deflection plates

Ultraviolet laser

Data analysisoscilloscope

amplifier

MALDI-TOF Schematic

An aliquot of this removed and mixed with a solution containing a vast excess of a matrix. sinapinic acid( protein analysis), ±-cyano-4-hydroxycinnamic acid(peptide analysis)An aliquot applied to the sample, allowed to dry prior to insertion into the high vacuum. laser is fired, the energy arriving at the sample/matrix surface optimized, and data accumulated as m/z spectrum

Tof analyzer separates ions according to their m/z ratios. The heavier ions are slower than the lighter ones.

Results in the generation of singly charged ions regardless of the molecular weight.

In +ve ionisation mode the protonated molecular ions (M+H+) are usually the dominant species. Positive ionisation is used in general for protein and peptide analyses.

In -ve ionisation mode the deprotonated molecular ions (M-H-) are usually the most abundant species. Negative ionisation can be used for the analysis of oligonucleotides and oligosaccharides.

Sample dissolved in an appropriate volatile solvent

Flow chart

For a particle of Mass = m and charge = z, accelerated through a potential V between plates distance d apart: How long t does it take to complete the trip? What is final speed v? What is the relationship between t (time-of flight) and M/z (mass to charge ratio)?

Theoretical Basis of TOF Separations

Theoretical Basis for TOF-MS

Charge = zAccelerating voltage = VMass = mVelocity = vDistance = dTOF = t

zV = ½ mv2

V = ½ mv2/z2V= mv2/zbut v = d/tm/z = [2V/d2]t2

t = (m/z)1/2(d2/V)1/2

Molecular Ion[Mṇ]⁺

Dimer[MṇMṃ]⁺

Trimer

Tetramer

Molecular weight Determination

Sample ionization methodsThe ionisation methods used for the majority of biochemical analyses are

Electrospray Ionisation (ESI)

Matrix Assisted Laser Desorption Ionisation (MALDI).

Other Ionisation methods include:

Atmospheric Pressure Chemical Ionisation (APCI)

Chemical Ionisation (CI)

Electron Impact (EI)

Fast Atom Bombardment (FAB)

Field Desorption / Field Ionisation (FD/FI)

Thermo spray Ionisation (TSP)

Ion source mass analyzer detector

Involves transfer of molecules into a vacuum without decomposing them.

Discovered- in the late 80's in the group of Prof. Fenn at Yale.

The intact transfer of large molecules from the liquid gas phase by an ion desorption mechanism, a direct emission of large molecules from liquid droplets.

Operate under atmospheric conditions.

Electrospray Ionization

Electrospray Ionization (ESI)

The sample solution is sprayed across a high potential difference (a few kilovolts) from a needle into an orifice in the interface.

Heat and gas flows are used to desolvate the ions existing in the sample solution.

Electrospray ionization can produce multiply charged ions with the number of charges tending to increase as the molecular weight increases.

The number of charges on a given ionic species must be determined by methods such as:

comparing two charge states that differ by one charge and solving simultaneous equations

looking for species that have the same charge but different adduct masses

examining the mass-to-charge ratios for resolved isotopic clusters

Electron Spray Ionization(ESI)(J. Fenn, J. Phys. Chem., 1984, 88, 4451)

Polar molecule analysis, molecules ranging from less than 100 Da to more than 1,000,000 Da in molecular weight.

ProcedureSample is dissolved in a polar, volatile solvent and pumped through a narrow, stainless steel capillary. High voltage applied to the tip of the capillary.Sample emerging from the tip is dispersed into an aerosol of highly charged droplets, aided by nebulising gas flowing around the outside of the capillary.Charged droplets diminish in size by solvent evaporation, assisted by a warm flow of nitrogen known as the drying gas which passes across the front of the ionisation source. Charged sample ions, free from solvent, are released from the droplets, pass through an orifice into an intermediate vacuum region, and from there into the analyzer of the mass spectrometer, under high vacuum.

Diagrammatic of Electronspray Ionization

Mechanism of Electronspray ionization

Original dropletContains + and – Ions; + predominant

+ + - +- + -- + +

- +

++ +

-+ + -

++

+

Solvent evaporatesField increases, andIons move towardsurface

As fieldincreases,ions areemitted fromdrop

In volatileresidue

The micro droplet shrinks due to solvent evaporation The resulting increase in charge density of the droplet, forces the charged analyte ion out of the solution before the droplet breaks up.

Sample introduction

Flow injection

LC/MS

Typical flow rates are less than 1 micro liter per minute up to about a milliliter per minute.

Benefits

Good for charged, polar or basic compounds

Permits the detection of high-mass compounds at mass-to-charge ratios that are easily determined by most mass spectrometers (m/z typically less than 2000 to 3000).

Best method for analyzing multiply charged compounds.

Very low chemical background leads to excellent detection limits.

Can control presence or absence of fragmentation by controlling the interface lens potentials.

Compatible with MS/MS methods.

Limitations

Multiply charged species require interpretation and mathematical transformation (can be difficult sometimes).

Complementary to APCI. Not good for uncharged, non-basic, low-polarity compounds (e.g. steroids).

Very sensitive to contaminants such as alkali metals or basic compounds.

Relatively low ion currents

Relatively complex hardware compared to other ion sources

Mass range

Low-high Typically less than 200,000 Da.

In positive ionization mode, a trace of formic acid is often added to aid protonation of the sample molecules.

In negative ionization mode a trace of ammonia solution or a volatile amine is added to aid deprotonation of the sample molecules.

Proteins and peptides are usually analyzed under positive ionisation conditions and saccharides and oligonucleotides under negative ionisation conditions.

In all cases, the m/z scale must be calibrated by analyzing a standard sample.

Positive or Negative Ionizsation? If the sample has functional groups that readily accept a proton (H+) then positive

ion detection is used e.g. amines R-NH2 + H+ ® R-NH3+ as in proteins, peptides

If the sample has functional groups that readily lose a proton then negative ion detection is used e.g. carboxylic acids R-CO2H ® R-CO2– and alcohols R-OH ® R-O– as in saccharides, oligonucleotides

Positive and negative Ion mode

In ESI, samples (M) up to 1200 Da give rise to singly charged molecular-related ions, usually protonated molecular ions of the formula (M+H)+ in positive ionisation mode and deprotonated molecular ions of the formula (M-H)- in negative ionisation mode.

Samples (M) with molecular weights > 1200 Da give rise to multiply charged molecular-related ions such as (M+nH)n+ in positive ionisation mode and (M-nH)n- in negative ionisation mode.

Proteins have many suitable sites for protonation as 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.

m/z = (MW + nH+)/ n

where m/z = the mass-to-charge ratio marked on the abscissa of the spectrum;

MW = the molecular weight of the sample n = the integer number of charges on the ions H = the mass of a proton = 1.008 Da.

Expression of m/z value

Leucine enkephalin

TEST0132(1.679)cm(3:34)

Platform II, BMB, University of Leeds

ESI-MS analysis of Leucine enkephalinCalculated MW.555.2 DaMeasured MW.555.1Da

4 Oct 199910:12:26

Scan ES+2.87 e5

M/Z Spectrum in positive ionization mode

The m/z spectrum also contains ions at m/z 578.1, some 23 Da higher than the expected molecular weight. These can be identified as the sodium adduct ions, (M+Na)+, and are quite common in electrospray ionization.

Interpretation

Electrospray ionization is known as a “soft” ionization method as the sample is ionised by the addition or removal of a proton, with very little extra energy remaining to cause fragmentation of the sample ions

By raising the voltage applied to the sampling cone, extra energy is supplied to the sample ions which can then fragment. The m/z spectrum then has extra peaks corresponding to sample fragment ions which can help in the structural elucidation of the sample.

Known as “cone voltage” or “in-source” fragmentation and although it can provide useful information it must be remembered that it is not specific so if there are a number of components in a sample, all will fragment to give rise to an extremely complicated spectrum.

Advantages of Multiple Charging

Can use instruments with lower maximum m/z (i.e., Quadrupoles, ion traps, FTMS)

For FTMS, the resolution is better at lower m/z values, therefore, ESI helps one obtain better resolution at higher m/z values.

Multiply charge ions tend to fragment easier then singly charge ions.

M/Z value expression

Platform II, BMB, University of Leeds

Hen egg Lysozyme

LYSO1A 1(1.392)Sm(SG, 2X1.00): Sb(10.10.00)

25 Jan 2000 10:10:37

ScanES+ 2.16e6

m/z = (MW + nH+)

n where

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

M/Z value expression

1431.6 = (MW + nH+) and 1301.4 = (MW + (n+1)H+) n (n+1)

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

n(1431.6) –nH+ = (n+1)1301.4 – (n+1)H+

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

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

and: n= (1301.4 - H+)

(1431.6 – 1301.4) hence the number of charges on

the ions at m/z 1431.6 = 1300.4 = 10. 130.2

1431.6 = (MW + nH+)n

gives 1431.6 x 10 = MW + (10 x

1.008) and so MW = 14,316 – 10.08 therefore MW = 14,305.9 Da

Peptide Mass Fingerprinting

peptides

Peptide mass fingerprint Peptide fragments

Peptide mass search MS/MS search

Schematic representation of MALDI-TOF mass spectrometer

MALDI ionization process MALDI-TOF operating in linear mode

MALDI-TOF instrument equipped with a reflection

Peptide Mass Fingerprinting

Effect of Mass accuracy and Mass Tolerance on peptide Mass Fingerprinting search result

Search m/z Mass tolerance #Hits

1529 1 478

1529.7 0.1 164

1529.73 0.01 25

1529.734 0.001 4

1529.7348 0.0001 2

Searches were done with the MS-FIT program

Effect of Multiple Peptide Masses on Protein Identification

Search m/z Mass tolerance #Hits

1529.73 0.1 204

1529.73

1529.7O 0.1 7

1529.73

1252.7O1833.88 0.1 1

Searches were done with the MS-FIT program

The Actual Peptide M/Z values are 1529.7348,1252.7074,1833.8845

Protein matches for peptide Mass Fingerprinting of m/z 1529.73

Peptide sequence identification Matched m/z

IGGHGAEYGAEALER Mouse Hb alpha 1529.7348

VGAHAGEYGAEALER Human Hb alpha 1529.7348

MGTGWEGMYRTLK Mouse lens epithelialCell protein LEP503

1529.7245

MADEEKLPPGWEK Human PINI-like protein

1529.7310

DTQTSITDSSAIYK Mouse signal recognition particle

1529.7335

NDSSPNPVYQPPSK Mouse peroxisome assembly factor-1

1529.7236

MNLSLNDAYDFVK Human dual specificity protein phosphatase 7

1529.7310

Interpretation of PMFExercise for participants

+

Carbamidomethylation 57 Da

Total 2188 Da

+

N-terminal Peptide Identified N-terminal Peptide Identified

Insilico digested N-terminal peptide for FILGRASTRIM

MTPLGPASSLPQFLIKCLEN-terminal peptide sequence

m/z=1095

MTPLGPASSLPQFLIKCLEN-terminal peptide sequence

m/z=1095

MTPLGPASSLPQFLIKCLEAbsence of N-terminal sequence

m/z=1095

MTPLGPASSLPQFLIKCLEAbsence of N-terminal sequence

m/z=1095

MTPLGPASSLPQFLIKCLEN-terminal peptide sequence

m/z=1095

MTPLGPASSLPQFLIKCLEN-terminal peptide sequence

m/z=1095

Schematic of Triple-Quad MS

Mass Changes from Post translational Modification

Mass Change Modification -2.0 Disulfide bond formation+14.0 Methylation+16.0 Hydroxylation+28.0 Formylation+30.0 Nitrosylation+42.0 Acetylation+80.0 Sulfation+80.1 Phosphorylation+180 Mono-glycosylation+204.4 Farnesylation+210.4 Myristoylation