Mass spectrometry

Tuula NymanProtein Chemistry Research GroupInstitute of Biotechnologytuula.nyman@helsinki.fi

Mass spectrometry is an analytical technique thatidentifies the chemical composition of a compoundor sample based on the mass-to-charge ratio ofcharged particles

Mass spectrometry

ION SOURCE: molecules of interest are ionized

MASS ANALYZER:ions are separated according to their m/z-ratios

DETECTOR: separated ions are detected

MS of small molecules

-In 1918, Arthur Jeffrey Dempster developed the first modernmass spectrometer, and established the basic theory anddesign of mass spectrometers that is still used to this day

-1919 Francis Aston constructs the first velocity focusing massspectrograph with mass resolving power of 130 (1922 NobelPrize in chemistry)

-The use of a mass spectrometer as the detector in gaschromatography was developed during the 1950s by RolandGohlke and Fred McLafferty

-Ionization modes: chemical ionization (CI) and electronionization (EI), not suitable for labile biomolecules

Biological mass spectrometry

-two modes of ionization: MALDI (matrix assisted laserdesorption ionization) and ESI (electrospray ionization)

developed in 1980’sNobel-price in Chemistry 2002

TAR

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PULSEDLASER

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MALDI= matrix assisted laserdesorption ionization

An ionization process suitable for mass spectrometric analysisof large molecules, like proteins and peptides. The analytesubstance is embedded in a crystallized matrix, which isirradiated by a laser. The power of the laser beam is usuallyadjusted in a way that it has enough energy to ionize thebiomolecules and matrix molecules but does not split thelarge analyte molecule. Thus MALDI belongs to the "soft"ionization techniques.

Commonly used matrixes for MALDI-TOF mass spectrometry

================peptides

===================proteins

A 384 position MALDI-TOF sample target plate. To each position 0,5-1µl ofsample together with matrix solution is pipetted and allowed to dry.

Electrospray ionization (ESI):Creation of ions by spraying a solution into an electrical field.This process, which belongs to the "soft" ionization techniques,enables the analysis of intact biomolecules, such as e.g. proteinsand peptides by mass spectrometry.

Electrospray of peptides in 0.1% FA /ACN from a 15 m I.D. fused silica glassneedle. The liquid flow is 200 nl/min and the needle has a potential of 2000V ascompared to the cone inlet of the mass spectrometer.

• sample is crystallized• produces mainly singly

charged ions• simple, easy-to-use• more tolerant to

salts+other contaminantsin the sample than ESI

• liquid sample• produces multiply

charged ions• easy to couple with HPLC

MALDI ESI

Mass analyser types

Time-of-flight mass spectrometry (TOFMS) is a method of massspectrometry in which ions are accelerated by an electric field of knownstrength. This acceleration results in an ion having the same kinetic energy asany other ion that has the same charge. The velocity of the ion depends on themass-to-charge ratio. The time that it subsequently takes for the particle toreach a detector at a known distance is measured.

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IONSOURCE

FLIGHTTUBE DETECTOR

Quadrupole mass analyzers use oscillating electrical fields to selectivelystabilize or destabilize ions passing through a radio frequency (RF) quadruple field.The quadrupole consists of four parallel metal rods. Each opposing rod pair isconnected together electrically and a radio frequency voltage is applied betweenone pair of rods, and the other. A direct current voltage is then superimposed onthe R.F. voltage. Ions travel down the quadrupole in between the rods. Only ionsof a certain m/z will reach the detector for a given ratio of voltages: other ionshave unstable trajectories and will collide with the rods. This allows selection of

a particular ion, or scanning by varying the voltages

MS in protein chemistry/proteomics/structural biology

-protein identification

-protein MW determination (NOT =identification!)

-characterisation of post-translational modifications

-relative quantification of proteins between samples

-analysis of protein complexes

-MS imaging

Protein MW determination-MALDI TOF MS, linear mode-accuracy is not as good with ESI MS

Singly charged ion

Doubly charged ion

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Protein MW determination, ESI MS

Deconvoluted electrospray mass spectrum of myoglobin

Protein identification methods:

•Peptide mass fingerprinting by MALDI TOF

•Partial sequencing by MS/MS

Protein identification by mass spectrometry

• protein of interest is cleaved into peptides witha specific enzyme

• peptides are analyzed by MS

SS

N

C

N

CAlkylation

K

R

KK

K

R

KRTrypsin

”In-gel” or ”in liquid” digestion of a protein

intact protein

Reduction

alkylated protein tryptic fragments from the protein

PEPTIDE MASS FINGERPRINT (PMF)

Gimenez spot 4

PEPTIDE MASS FINGERPRINTING

• requires a very spesific enzyme• optimized digestion+ desalting protocols• internal/close external calibration of MALDI spectra• works only for proteins which are already in the

databases as protein sequences

Tandem mass spectrometry

C H I L PLH

IIL

Inte

nsity

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nts

m/z, amu

TOF product 745,4

MS scan:

MS/MS scan:Peptides with certain m/z-ratio areselected and fragmented inside the

mass analyzer, and the m/z-ratios of thefragment ions are measured

-both MW and sequence information from the peptides in one experiment-mass analysers: triple quad, ion trap, Q-TOF, TOF-TOF

The ions are first separatedaccording to their m/z ratios

Protein identification usingProtein identification using nanoLCnanoLC--MS/MSMS/MS

-75 um i.d. columns, 200 nl/minno need to split the effluent before MS

-DDA= data dependent analysis-can be fully automated-suitable for complex protein mixtures, possibility toidentify hundreds of proteins in one run-requires efficient data processing tools

-different database search programs can producedifferent results from the same raw data-false positive rate estimation

What Follows ’Simple’ Protein Identification?

• Defining N- and C-termini• Classification of splice variants• Characterization of protein modifications

Protein Characterization

Protein Modifications• Identification of modified protein• Localization of modification• Structure elucidation of the modification

The second level of proteome analysis- analysis of modified proteins

Some of the most common modifications

•• NN--terminusterminus•• LysineLysine

AcylationAcylation•• SpecificSpecific•• NonNon--specificspecific•• CC-- and/or Nand/or N--terminalterminal

ProteolyticProteolytic processingprocessing

•• OO--linked (Serine,linked (Serine, ThreonineThreonine, ...), ...)•• NN--linked (linked (AsparagineAsparagine))

GlycosylationGlycosylation

PhosphorylationPhosphorylation•• SerineSerine•• ThreonineThreonine•• TyrosineTyrosine•• (Aspartic acid,(Aspartic acid, histidinehistidine andand

lysine)lysine)

““GlycoGlyco--lipidslipids””•• GlycosylGlycosyl--phosphatidylinositolphosphatidylinositol anchoranchor•• FarnesylFarnesyl anchoranchor

•Specific detection in gelsRadiolabelingFluorescent labelingWestern blottingModification specific stains

•Affinity fishingImmune precipitation of proteinsAffinity purification of proteins/peptides

-phosphopeptide isolation: IMAC, TiO2-phosphoprotein isolation: phosphospecific antibodies

Selective tagging followed by affinity purification

•Selective mass spectrometryPrecursor ion scanningNeutral loss scanningStable isotope labeling

Analysis of modified proteins

Identification of EGF signaling moleculesby immunoprecipitation and mass spectrometry

Immunoprecipitation with anti-phosphotyrosine antibody

•EGF Receptor

•Hrs*

•Cbl*

•Eps15

•p62

•Shc

•p85 subunit of PI 3-Kinase

•SHIP-2

•STAM*

•Vav-2

•STAM2*

•Odin

•Ku 70 Autoantigen

•Hsp 70

Pandey et al (2000) PNAS 97 179-184*Steen et al (2002) J. Biol. Chem. 277 1031-9.

D.

Phosphopeptides enriched using IMAC

followed by sequencingby ESI-MS/MS

A. Stensballe & O.N. Jensen

MALDI mass map of in-geldigested protein (CK2)

Verification of phosphorylation

Cells/tissues

Protein separation (2-DE)

Imaging and relative quantitation(=gel and protein comparison)

Protein spots of interest are cut fromthe gel and protein's are in-gel digested

Gel-based proteomics

Mass spectrometric analysis ofthe resulting peptides(nanoLC-ESI-MS/MS or MALDI-TOF/TOF)

Mixing of the samples and in-solution digestion

Protein extraction from thesamples

Cells/tissues

protein labelling (35S, 32P, CyDye)

B) protein labelling (e.g. ICAT)

Protein extraction from the samples

A) protein labelling (SIL, 15N, 13C)

Identification of proteins of interestDatabase searches with MS and MS/MS-data

MS-based proteomics

Chromatrographic peptide fractionation

Mass spectrometric analysis of peptide fractions(nanoLC-ESI-MS/MS and nanoLC-MALDI-MS/MS)

Relative quantitation based ondifferentially labeled peptides

Two-dimensional electrophoresis

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pI 103

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-15

Proteins are separated accordingto their pI and molecular weight

Every sample is run on it's owngel

For identification, spots ofinterest are cut out from the gel,in-gel digested, and the resultingpeptides are analysed with MSand database searches

•20 x 106 Th cells•35S-Met labelling (24 h)•18 cm 3-10 NL IPG-strip•12% SDS-PAGE

Human macrophages infected with Influenza A virus,Cytoplasmic proteomes

-For identification, the protein spots are cut out from the gel, in-gel digestedinto peptides and identified with MS and database searches-Works with low-femtomolar amounts of proteins

•2-DE is an efficient method to separate very complexprotein mixtures

•2-DE separates also protein isoforms into distinctspots

•certain protein classes, e.g. very big or small proteinsand proteins with extreme pI:s are absent orunderrepresented in 2-DE gels

•a lot of manual lab work

2-DE is a good separation method but…

MS-based proteomics

•MS-based proteomics hasshown to be an efficienttool for e.g. membraneprotein analysis

•for quantificatication theproteins/peptides need tobe labeled

•labeling is usually donewith stable isotopes

•peptide separation aftertrypsin digestion usingdifferent forms of LC

Isotope-coded affinity tag (ICAT) technique

• ICAT technique was introduced by Gygi et al. (1999)

• ICAT is an approach for quantification and identification ofindividual proteins within complex mixtures

- quantification is based on stable isotope labeling

• ICAT technique makes it possible to study some classes ofproteins which are excluded or underrepresented in 2-DE gels

- Membrane proteins

- Large and small proteins

- Extremely acidic and basic proteins

- Low-abundance proteins

• Possibility to automate many steps of the method fromsample preparation to data analysis

First generationICAT reagent

Cleavable ICAT reagents

Other stabile isotope labels

N-terminus Nic-NHS (H/D)Phenyl isocyanate (H/D)

Munchbach et al. 2000Mason et al. 2003

C-terminus Trypsin digestion (16O/18O) Yao et al. 2001

N- and C-terminus

X3-N-acetoxysuccinamide(X=H/D)

Liu & Regnier 2002

Cysteine Acrylamide (H/D) Secchi et al. 2002

Tryptophan 2-Nitrobenzenesulfenyl(12C/13C)

Kuyama et al. 2003

Lysine O-methylisourea Cagney & Emili 2002

Histidine N-acetoxy-X3-succinimide(H/D)

Wang et al. 2002

Phospho-protein

-SCX2CX2S- (X=H/D) Goshe et al. 2001 and2002

ICAT method

Protein identification and quantification in ICAT

C H I L PLH

IIL

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nts

m/z, amu

TOF product 745,4

Inte

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nts

m/z, amu

TOF MS 740-750 amu

Identification fromMS/MS data

Quantificationfrom MS data

HeavyLight

MS spectrum

MS/MS spectrum

The ICATThe ICAT TechniqueTechnique

• Cysteine specific– Simplifies mixtures into cysteine containing peptides only– Complexity down to around 10-20 %

– Cysteine in 80-90% of all proteins only.– Incomplete proteome coverage– Loss of PTM information

Reporter groupMass 114-117

Balancing groupMass 28-31

Peptide reactive group:binds covalently to each

lysine side chain andN-terminal group of

a peptide

Isobaric tagTotal mass 145

LC-MS

114115

116

117

LC-MS/MS

m/zt

iTRAQ = isotope tagged relative and absolutequantitation

iTRAQ workflow

Isolated protein pellets

Protein reduction, alkylation, and in-solution digestion

iTRAQ labelling of the peptides

Pooling of the labelled samples

SCX fractionation of the peptides

LC-MS/MS analysis for the SCX fractions

Protein ID and quantification based on MS/MS data

~20 peptide containing SCX-fractions, every fraction isanalysed separately by LC-MS/MS

Exp 1, TOF scan

Exp 2, product ion scan

Exp 3, product ion scan

38584152downregulated(fold difference < 0,67)

370175295upregulated(fold difference > 1,5)

10269471455proteins identified

Nuclearfraction

Cytoplasmicfraction

Mitochondrialfraction

5,9714,0086,4919,3107,2851,8687,1767,2943,005Hemagglutinin precursor - Influenza Avirus

5,52912,29310,9637,37710,8442,72916,6178,1692,415Matrix protein 1 - Influenza A virus

0,6950,7120,88011,8198,6951,1931,2130,3831,151Histone H4 - Homo sapiens

0,7830,7040,84215,7929,5550,7161,2710,4101,285Histone H2A.x - Homo sapiens

3,3093,0261,9841,9121,0211,0556,4023,2602,424ANXA5 protein (Fragment) - Homosapiens

1,7021,2271,3881,8541,1741,5012,5231,4971,534Annexin A2 - Homo sapiens

8,0914,1341,6571,4581,0831,1075,4462,7781,947Annexin A1 - Homo sapiens

117:114116:114115:114117:114116:114115:114117:114116:114115:114Name

nuclearcytoplasmicmitochondrial

iTRAQ to characterise influenza A virus infected human macrophages

Analysis of protein complexes using mass spectrometry

8, 645-654 (August 2007)

Tandem affinity purification (TAP): decreased background levels

Isobaric tags to elucidate complex formation dynamics

iTRAQ

Advantages:

• Not cysteine specific, labels every peptide

• Retains greater proportion of information of PTMs

• More peptides for confident identification

• Quadraplex: Four comparisons at the same time

• Can use 3 labels for 3 different systems plus the 4th asan internal standard for absolute quantification

• Labelled peptides isobaric: MS/MS fragmentationinformation overlaid in the same m/z window,enhancing identification

ICAT and iTRAQ

ICAT: labelling at the protein level (before digestion)quantification based on MS dataidentification based on MS/MS data

iTRAQ: labelling at the peptide level (after digestion)both ID and quantification based on MS/MS data

BOTH produce huge amounts of raw datacurrent bottlenecks are in data analysis andvalidation of the results

• automation possible (very little manual lab work)

• possible to study all protein classes

• data validation is challenging