Karen Jonscher, PhDDirector, SBCF/TATC Proteomics Core FacilityUCD, School of Medicine, Department of Anesthesiology

BS in Engineering Physics, CU Boulder MS, PhD in Applied Physics, Caltech Thesis work – building nanospray and MALDI

sources as well as hybrid quadrupole ion trap MS, Lee Hood/John Yates

PostDoc, National Jewish, phosphoprotein analysis CU Boulder, Katheryn Resing, methods

development for shotgun proteomics, data validation

UCD, Associate Professor, Proteomics Core Director, SBCF/TATC Mass Spectrometry Core Facility

Identify the elements of the proteomics workflowSample preparation basics Separations HPLC Affinity

Mass spectrometry basics Ionization Elements of a spectrum Isotopes Information content

How can mass spec be used?

Inlet Ion source Mass analyzer Detector Software

What is my protein (i.e. protein ID)? How does the amount of my protein(s)

change in a healthy cell vs. a diseased cell (or tissue or blood or…)?

Does my protein interact with other proteins? Do any chemical changes happen to my

protein that might affect how it functions?

We try to answer these questions using Mass Spectrometry (multiplexed Ab/aptamerarrays are also emerging)

Mass Spectrometers measure the molecular weight of ionized molecules

Baseline Hypothesis: Use the weight of pieces of protein to figure out the protein identity/modifications

M-P-G-R-P-G-G-P-A-V…

20 common amino acids with different side chains that have different chemical properties

>sp|P68871|HBB_HUMAN Hemoglobin beta subunit (Hemoglobin beta chain) (Beta-globin) - Homo sapiens (Human). VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

Hemoglobin has 2 alpha and 2 beta chains. These chains interact and form a 4 unit structure.

Sickle Cell Anemia results when valine is substituted for glutamic acid in both beta chains. This causes the mutant hemoglobins to stick to other mutant hemoglobins, forming a polymer. The result is misshapen cells that cannot transfer oxygen through small blood vessels.

Sickle Cell Normal CellAffects transport of oxygen to organs and muscles – leads to

pain, anemia (weakness), infection, sometimes death.

Sample Preparation

Mass Spectrometric Analysis

Data Analysis and Mining

Separation Fractionate by solubility (cytoplasmic/nuclear) Fractionate by organelle (mitochondria, Golgi,

etc) Fractionate by protein (size, charge, affinity)

Enzymatic digestion Separation Fractionate by peptide (size, charge)

The Importance of Separation

Peptide level

Protein level

Due to signal suppression, mass spectrometers can only analyze a limited number of ions at a time

Gel-based approach

Use polyacrylamide gels, isoelectric focusing to separate proteins

Use HPLC to simplify complex mixtures Size (gel filtration, reverse phase)

Affinity (immunodepletion)

Hydrophobicity (reverse phase)

Basicity (cation/anion exchange, SCX)

Concentration 10% 20% 40% 60%

422.0607.5

842.8

0.00.51.01.52.02.5

6x10

Intens.

400 600 800 1000 1200 1400 1600 1800 2000 m/z

594.6

736.6

798.6

899.0

963.61020.6

1141.6

1402.8012345x10

Intens.

400 600 800 1000 1200 1400 1600 1800 2000 m/z

Low Abundance Proteins

High Abundance Protein

flow through

Further processing and MS

elute

Flow Through(Low Abundance)

Eluate(High

Abundance)

Fractionate the fractions!

High Abundance Flowthru 1 Flowthru 2

Albumin288

Albumin0

Albumin0

FT.D: BPC 100-2200 +All MS

0.0

0.5

1.0

1.5

2.0

8x10Intens.

20 40 60 80 100 120 Time [min]

Use information from gel as well as MS data to identify proteins

Need to have multiple hits to identify proteins.

Shotgun ProteomicsMulti-Dimensional LCMudPIT

2DGEDIGE

The sample (fractionated/immunodepleted/immuno-enriched) is ready for proteomics – now what?

2D GELS Visualize

hundreds/thousands of proteins

Quantitation by densitometry – stain

Easy to observe PTMs Not good for membrane,

large, small, basic, acidic protein

CHROMATOGRAPHY Get list of

hundreds/thousands of IDs Quantitation by labeling –

iTRAQ, SILAC - or spectral count

PTM detection dependent upon sequence coverage

Protein coverage may be low – rely on statistical methods for ID

Ion Source Mass Analyzer Detector

ESI

MALDI

EI

CIAPCI

APPI

DIOS

FAB

LSIMS

Fourier Transform

Time-of-Flight

Ion Trap

Quadrupole

Electron Multiplier

Multichannel Plate

Photomultiplier

Inlet

HPLC

Probe

Plate

Syringe

GC

Vial

Ion Source Mass Analyzer Detector

ESI

MALDI

Fourier Transform

Time-of-Flight

Ion Trap

Quadrupole

Electron Multiplier

Multichannel Plate

Photomultiplier

Inlet

HPLC

Plate

NSI

1. An ion is an electrically charged molecule.

2. An ion can be positively (+) charged or negatively (-) charged.Consider the poles on a battery.

3. Molecules must be charged to be measured by a mass spectrometer.

4. A mass spectrometer "weighs" molecules electronically by attracting and repelling ions. Consider magnets. Opposites attract. Like charges repel.

- +

Removing an electron (EI/CI)Adding a proton (ESI/NSI/MALDI)

Adding a cation (Na)

Removing a proton (negative ions)

Capturing an electron (EC)

Charged Droplet

Stainless steel capillary50 mm fused-silica capillary

MS

HV (-ve)

+ ++ ++

++ + +

+

Taylor Cone

Monodisperse drops

• extremely gentle ionization technique• generates multiply-charged ions• ideal for macromolecules

atmosphere

5-20 ml/min

3-4 kV

N2

MW = molecular weight of the targetN = integer number of charges on the ionH = mass of a proton (1.007825 Da) providing the charge

Resources for nanospray at www.newobjective.com

10-400 nl/min flow rate Droplets many times smaller than

conventional ESI 2-3 times higher ion current than ESI

at a given concentration Smaller tip close to inlet – better ion

transfer No nebulization 100X improvement in efficiency Works for high polarity solvents like

pure water Tolerates salts better than ESI Static vs. dynamic nanospray Pioneering work: Wilm and Mann,

Anal Chem, 68 (1996) 1-8.New Objective

Peptides, proteins, oligonucleotides, carbohydrates, natural products, lipids

H+ Uex

Mass Analyzer

MatrixCation (e.g. Na+)AnalyteNa+

Laser – 337 nm

Extraction Voltage – 25 kV

Isolate and encase the analyte molecules (analogous to a solvent shell) Absorb the laser energy via electronic or vibrational coupling/excitation Facile desorption from the condensed phase WITH the analyte

molecules but WITHOUT destructive heating of the analyte molecules (“softness”)

Efficient ionization of analyte molecules

Karas et al. J. Mass Spectrom. 2000, 35, 1-12.K. Dreisewerd Chem. Rev. 2003, 103, 395-425

= Analyte

= Matrix

Photons= “Excited” Matrix

MALDI

Electrospray

1000 1500 2000m/z

100

75

50

25

066,000 67,000

Molecular Weight

reconstruct(%)

66,400

30+32+

34+

36+

38+

41+45+

47+

49+

51+

53+

bovine serum albumin

Mass Range vs. m/z range

ESI Benefits ESI Detractors

MALDI Benefits

MALDI Detractors

Multiply chargedMultiply charged ions difficult to

interpretSingly charged Singly charged ions

difficult to dissociate

Salts disrupt signal

Tolerate salts

No matrix interferenceMatrix causes

background at m/z <700

Can interface directly to LC

Slow analytical throughput

Quick analytical throughput

Signal suppression limits mixture complexity

Ion Source Mass Analyzer DetectorInlet

A mass spectrometer is used to help scientists:

1. Identify molecules present in solids, liquids, and gases

2. Determine the quantity of each type of molecule.3. Determine which atoms comprise a molecule and

how they are arranged

500 1000 1500 2000 2500m/z

Rel

ativ

e Abu

ndan

ce The number of components in a sample The mass-to-charge of each component in a sample The relative abundance of each species The structure of each component in a sample

ionization

mass analysis

detection

10-4 – 10-9 torr

Atm – 10-6 torr 10-6 – 10-9 torr

m/z

Animated graphic courtesy of ionsource.com

Separating the ions

•Pocket change (mixture of coins)•Penny, dime, nickel, quarter, half $•Sorting change by value or size

•Mixture of molecules•Molecules of different weight, size•Separation by mass spectrum

3

8

5

4

2

dime penny nickel quarter half $

Size (m/z)

Qua

ntit

y (A

bund

ance

)

From Gross

m/z 12.0000 -> C, 1 u = 1/12 12C1 u increments -> loss of H12 + 4 = 16 = C + 4H = CH4

Exact mass = 46.04Full mass = 46.07?

StandardAtomic Weight

IsotopicComposition

RelativeAtomic MassIsotope

Coursey, J. S., Schwab, D. J., and Dragoset, R. A. (2001). Atomic Weights and Isotopic Compositions(version 2.3), [Online]. Available: http://physics.nist.gov/Comp [2002, April 22]. National Institute of Standards and Technology, Gaithersburg, MD.

0.205(14) 17.999 160 4(9) 18

0.038(1) 16.999 131 50(22) 17

15.9994(3) 99.757(16) 15.994 914 6221(15) 16 O

N

0.368(7) 15.000 108 8984(9) 15

14.0067(2) 99.632(7) 14.003 074 0052(9) 14

14.003 241 988(4) 14

1.07(8) 13.003 354 8378(10) 13

12.0107(8) 98.93(8) 12.000 000 0(0) 12 C

http://www.ionsource.com/tutorial/isotopes

Resolving power => Ability of a mass spectrometer to distinguish between ions of different mass-to-charge ratios.

R=M/∆M M=m/z ∆M=full width of the peak at half

maximum Mass accuracy ∆M/M

500/1=500

Blue: ∆m = 3.48 R = 1000, 1000 ppmRed: ∆m = 1.161 R = 3000, 333 ppmGreen: ∆m = 0.348 R = 10,000, 99 ppmBlack: ∆m = 0.116 R = 30,000, 33 ppm

R = m/∆m

Where a monoisotopic peak can be identified, mass accuracy follows resolution. For some instruments, increased

resolution comes at the price of sensitivity.http://www.matrixscience.com/help/mass_accuracy_help.html

For a compound with a mass of 1000 Daltons:

1000 ± 2.0 Da (or ± 2000 ppm)

1000 ± 0.5 Da (or ± 500 ppm)

1000 ± 0.1 Da (or ± 100 ppm)

1000 ± 0.01 Da (or ± 10 ppm)

1000 ± 0.002 Da (or ± 2 ppm)

ESI-TOF

MALDI-TOF

3D Trap

Mass Accuracy # of Candidate Proteins in DB

1 Da 1000

0.1 Da 100

0.01 Da 10

Consider the peptide with sequence EAGPEMADQK

In proteomics, it’s all about the database search

MS ResolutionScan

SpeedMass

AccuracySensitivity

Quadrupole$-$$

+-+++ + +-+++ +

Ion Trap$

++ ++ + ++

TOF$-$$$

+ - +++ +++ +++ +++

FT$$$$

++++ + ++++ ++++

Two mass spectrometers joined by a chamber that breaks apart molecules.This definition is appropriate for tandem-in-space but not for tandem-in-time.

Puzzle Analogy

MS #1Fragmentation

Chamber MS #2

Sorting molecules

Breaking molecules Sorting pieces

1

1

2

1

2

1

Words

Comprised of letters.

Arrangement of letters gives

words meaning.

Special groups of letters make

syllables.

Common endings = suffixes.Talking

Writing

Playing

Use a computer to search for the string "ing" and it displays all words containing "ing“ in a certain document.

Molecules

Comprised of atoms.

Arrangement of atoms gives

molecules function.

Special groups of atoms

make functional groups.

Common side chains = esters.

H2NCH2COOC4H9

H2NCH(CH2)COOC4H9

H2NCH(C6H5)COOC4H9

Use a NL scan function to detect only molecules in your plasma sample that lose a butylformate function group weighing 102 Da

-2 kV

Signal

15 kV

Electron Multiplier

Conversion Dynode

MicrochannelPlate

Photocathode

Scintillator

Anode

Photomultiplier

A. High sensitivity• Ability to detect very small amounts

B. High selectivity• Ability to tell molecules apart in a mixture

C. High time resolutionD. Low CostE. I have no idea

Metals, Glass, Ceramics and Composites

Electronic Materials and Devices

Energy System Diagnostics: Solar, Fossil, Fission and Fusion

On-Line Monitoring and Process Control

Air and Water Monitoring

Agriculture and Food Science

Defining grain boundaries at ppm concentrations

Ion beam milling (0.2 mm) and etching

Coal and Petroleum Research

Food and brewing industry

Biological Warfare Detection, Spacecraft

Herbicides, insecticides and fungicides

a doctor, a lawyer and a mass spectrometrist

Magnetic Resonance Imaging (NMR)

Positron Emission Tomography

Hair

SalivaSputum

Breath

Blood

UrineVaginal FluidExpressed Prostatic Fluid

Feces (Meconium)

Lung

Heart

Brain

Fatty Tissue Plasma

Organs

CSF

Synovial Fluid

SampleIntroduction

Mass Analyzer

IonizationMethod

Direct InfusionDirect Probe

GCGC/GC

LCCE

2DLC

EICI

ESIMALDIPDMS

Spark SourceICP

Laser MicroprobeSIMSFAB

Field IonizationPyrolysis

ThermosprayAPCIAPPIDESI

DARTMALDESI

l-TOFr-TOF

l-QuadQIT

Magnetic SectorFT-ICR

Orbitrap

Tandem-MS(in-space)

QQQQIT-TOFQ-FTICRTOF-TOF

(in-time)FTICR

QIT

Problem

Identify the elements of the proteomics workflowSample preparation basics Separations HPLC Affinity

Mass spectrometry basics Ionization Elements of a spectrum Isotopes Information content

How can mass spec be used?

Inlet Ion source Mass analyzer Detector Software