Mass Spectrometry and Proteomics

Paolo Lecchi, PhDDept. of Pharmacology

George Washington University

October 13, 2003

WHAT IS A “MASS SPECTROMETER ”...?

MS

The black box problem…...

ESI MALDI LC/MS LC-MSMSESI

ESI Ion trap TOF QTOFquad.

ESI CRIMS EI APCIFAB

FT-ICR

qQTOF

SELDI

…many black boxes !

“A MASS SPECTROMETER MEASURES THE MW….”

“...A MS ANALYSIS GIVES THE MASS-TO-CHARGE RATIO (m/z)

OF IONS…IN GAS PHASE”.

Data Processing

ION SOURCE

ANALYZER ion separation

vacuum

Detector

Pumping system

Sample introduction

DIRECT INTRODUCTION

(solid, liquid, gas) SEPARATION

TECHNIQUE (HPLC, CE, GC)

MALDI, FAB, EI, ELECTROSPRAY

TOF, quadrupole, ION TRAP

ESILiquid flow

Q or Ion Trapanalyzer

ESI is a solution technique that gives a continuous stream of ions, best for quadrupoles, ion traps, etc.

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MALDI3 nS LASER PULSE

Sample (solid) on target at high voltage/ high vacuum

MALDI is a solid-state technique that gives ions in pulses, best suited to time-of-flight MS.

TOF analyzer

Atmosphere Low vac. High vac.

High vacuum

….MALDI or Electrospray ?

MALDI is limited to solid state, ESI to liquid

ESI is better for the analysis of complex mixture as it is directly interfaced to a separation techniques (i.e. HPLC or CE)

Until recently only ESI was available for high quality tandem-MS

MALDI is easier to use and maintain

MALDI is more “flexible” (MW from 200 to 400,000 Da)

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%Int.

5000 10000 15000 20000Mass/Charge

Data: 1015pl30001.O7 15 Oct 2002 16:22 Cal: 16 Oct 2002 8:12

8480.6

5653.4

16953.04239.4

MALDI-TOF spectra of apomyoglobin

INSTRUMENT: Kratos Axima-CFR

Sample: 1 pmole apomyoglobin (horse skeletal muscle)

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m/z

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+101695.9

+111541.8

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+141211.7

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1131.0

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1060.4

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998.1

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942.8

+19893.2

+20848.6

+21808.2

+22771.5

+23738.0+24

707.4+25

679.1

+91884.5

ACTUAL SPECTRUM

+91884.3

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mass

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16951.5

AFTER DECONVOLUTION

ESI-ion trap spectra of apomyoglobin

INSTRUMENT: Thermoquest LCQ-classic

Sample: 1 pmole apomyoglobin (horse skeletal muscle)

A “RESEARCH GRADE” MS (200 to 500 k$) PROVIDES AN ACCURATE MW DETERMINATION:

~ 10 ppm (e.g. 1000.0 ± 0.1)

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ACCURACY IS NOT THE ONLY PARAMETER TO BE CONSIDERED IN A MASS

SPECTROMETER...

...don’t forget the “S factors”

Sensitivity = femtomole 10-15 M (...attomole 10-18 M)

Simplicity = very easy training required

$$$ = 70 to 650 k$ 120 to 650 k$

Speed (high throughput)= ~104/day dynamic system

Structural information = MS/MS MSn

Software = “ ...evaluation in progress.”

MALDI ESI

Selectivity (resolution) = >5000

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2092 2094 2096 2098 2100 2102 2104 2106 2108Mass/Charge

Data: NDR_thin_2Ghz_blk_6fload0001.G24 20 May 2002 16:49 Cal: Kent_SP2_blk_1567 20 May 2002 14:31 Kratos PC Axima CFR V2.3.0a: Mode reflectron_2GHz, Power: 48, Blanked, P.Ext. @ 2095 (bin 135)

2099.17

2098.18

2100.18

2101.19

MALDI analysis of a peptide m/z 2098 (6 fmoles loaded)

Expected peptide mass 2098.20

(0.02 amu difference) Accuracy: (~10 ppm)

0.1 amu

resolution: ~20,000

NEW INSTRUMENT DESIGNS MAKE ESI AND MALDI MORE “INTERCHANGEABLE”...

Orthogonal acceleration-TOF is compatible with a continuous ion current, such as from ESI.

Liquid

Pusher electrode with pulsed VAcc

To reflectron TOF

Needle@4kV ions

N2 Laser

Ar collision gas

Hinged door

96-well MALDI target

1x10-2 Torr

2x10-5 Torr

5x10-7

Torr

Liner

Ion mirror

MCP

PusherPuller

Q1 q2

MS1 mass

selection

CID

MS2 mass selection

Thermal equilib.

q

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UV laser pulses via attenuator,

fiber optic cable and lens to

vacuum system

Schematic of QSTAR (with MALDI source)

MALDI can be carried out at “high” pressure. Ions collide with gas molecules, are slowed down and thermally equilibrated. Pulses are “smeared out” to give a semi-continuous ion current.

“...for their developments of soft desorption ionisation methods for mass spectrometric analysis of biological macromolecules”.

Nobel Prize in Chemistry 2002

1//2 of the prize went to Kurt Wutrich (Switzerland) development of NMR analysis

1/4 to John B. Fenn (USA)

Virginia Commonwealth University

1/4 to Koichi Tanaka (Japan)

Shimadzu. Corp. Kyoto

Electrospray Laser Ionization

“..other than biochemistry done very, very fast, is not entirely clear to me what proteomics is.”

Marvin Cassman, Director of the National Institute of General Medical Sciences *

C&E News, March 18, 2002

(*) ~90% of the NIH-funded mass spectrometry lab receive grants from NIGMS.

DIFFERENTIAL EXPRESSION AND QUANTITATION

THE ROLE OF MASS SPECTROMETRY IN PROTEOMICS:

IDENTIFICATION OF PROTEINS, USUALLY IN COMPLEX MIXTURES

ANALYSIS OF POSTRANSLATIONAL AND CHEMICAL MODIFICATIONS

Run 2Dgel; stain;

scan

Excise spot;elute; digest

1000 1500 2000 Mass (m/z)

Extract peptides;mass analyze

Protein identification

2D-PAGE AND MASS PECTROMETRY...…A PARADIGM IN PROTEOMICS

SEPARATION electrophoresis (1-D, 2-D)

chromatography (SEC, ion exchange, reversed phase)

DIGESTION chemical (BrCN)

enzymatic (trypsin,, Lys-C, Asp-C) reduction (Di-Thio-Threitol, -Mercapto-Ethanol) alkylation (IodoAcAcid, IodoAcAmide, Vynil Pyridine)

MALDI MS ANALYSIS

protein identification (peptide mass fingerprinting)

peptide structural information (post source decay)

EXPERIMENTAL PROCEDURES IN PROTEOMICS

SAMPLE CLEAN UP chromatography (reversed phase)

solid phase extraction (Zip Tip)

Mass spectrometry improved substantially during the last 10 years...

2D-PAGE still is the most powerful separation technique but has several disadvantages...

2-D PAGE AND MASS SPECTROMETRY... ...NOT THE IDEAL TECHNIQUE FOR PROTEOMICS:

Restricted to proteins < 106 and > 104 Da MW Cannot detect proteins expressed at low levels Limited to 600~800 separate spots Gel to gel reproducibility is poor Quantitation is poor, ± 50% or worse Dynamic range is limited, < 10X Analysis is not directly coupled to separation

DISADVANTAGES OF 2-D PAGE...

MULTI-DIMENSIONAL SEPARATIONS: AN ALTERNATIVE TO 2-D

PAGEIn multidimensional chromatography two (or more) techniques with “orthogonal” properties are combined to achieve higher separation power.

FIRST DIMENSION: e.g. Size Exclusion, Ion-Exchange

SECOND DIMENSION: e.g. Reversed Phase Ion-Exchange

MS

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RETENTION TIME

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0.34|-|0.37

0.31|-|0.34

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HYBRID 2-D SEPARATION: IEF-HPLC

Lecchi et. al. JBBM june 2003

Sample: E. coli extract

First dimension: y-axes IEF (Biorad rotofor)

Second dimension: x-axes IEF fractions separated by HPLC (reversed phase C-18)

…to improve the efficiency of the proteolytic digestion it is important to reduce and alkylate disulfide bonds

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RR

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reduction alkylation

enzymatic digestion

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Enzymatic digestion

*Some proteolytic enzymes are very specific, e.g.:trypsin cuts only at Lys-X or Arg-XLys-C Lys-XArg-C Arg-XGlu-C (V-8) Glu-X and Asp-X

peptide bond

R-C-OH

O

H2OProteolytic*

enzyme

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R-C N-R’

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N-R’

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protein

(proteolytic fragments)

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Mass/Charge

10 mV Profiles 1-50 Smooth Sv-Gl 2 -Baseline 20

Kratos PC Axima CFRplus V2.3.0: Mode reflectron, Power: 50, P.Ext. @ 1500 (bin 157)

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Peptide Mass Fingerprinting (PMF)

MS analysis of proteolytic fragments is a common way to identify a protein.

Low MW peaks (e.g. < 500 Da) are not generally used because of the high

interference of the matrix.

The following masses are entered in a database for protein identification: 737.99 - 874.44 - 936.48 - 1030.65 - 1047.06 - 1153.67 - 1269.76 - 1428.85 1536.76 - 1676.97 - 1808.06 - 1862.99 - 2163.26 - 2274.28

Database search for protein identification

“IS NOT JUST ABOUT ‘SEPARATION POWER”

AN IDEAL METHOD SHOULD BE ABLE TO IDENTIFY AND QUANTIFY PROTEINS WHOSE EXPRESSION LEVELS CHANGE

GENES 35,000

PROTEINS 200,000

TRYPTIC PEPTIDES 4,000,000

THERE IS A HUGE DYNAMIC RANGE OF PROTEIN EXPRESSION (12 orders of magnitude)

1 peptide/sec = ~46 days

“Proteomics is knowing the structure and function of all proteins from all organisms…that is not possible. We need to be more selective”.

George Kenyon, University of Michigan.

In his opening remark at the meeting: “Defining the Mandate of Proteomics in the Post-Genomic Era”.

National Academies, Washington DC, March 2002.