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Tuula NymanInstitute of Biotechnologytuula.nyman@helsinki.fi

Biological Mass spectrometryin Protein Chemistry

ION SOURCE: molecules of interest are ionized

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

DETECTOR: separated ions are detected

MASS SPECTROMETRYis an analytical technique that identifies thechemical composition of a compound or samplebased on the mass-to-charge ratio of chargedparticles

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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 laser desorption ionization)and ESI (electrospray ionization)

-developed in 1980’s by Michael Karas & FranzHillenkamp, Koichi Tanaka, John Fenn & MatthiasMann, Peter Roespstorff…

Nobel-price in Chemistry 2002 to Tanaka and Fenn’For their development of soft desorption ionisationmethods for mass spectrometric analyses ofbiological macromolecules’

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TAR

GE

TPULSEDLASER

+

++

+ +

++

+20 kV

MALDI= matrix assisted laser desorption ionization

The analyte substance is embedded in acrystallized matrix, which is irradiated by alaser. The power of the laser beam is usuallyadjusted in a way that it has enough energy toionize the biomolecules and matrix moleculesbut does not split the large analyte molecule.

A 384 position MALDI-TOFsample target plate.

Commonly used matrixes for MALDI-TOF mass spectrometry

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

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

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

Electrospray of peptides in 0.1% FA/ACN from a 15 um I.D. fused silicaglass needle. The liquid flow is 200nl/min and the needle has a potentialof 2000V as compared to the coneinlet of the mass spectrometer.

Mass analyser types

+orbitrap

All mass analyzers work in high vacuum

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Common MS-instrument types:-MALDI-TOF and MALDI-TOF/TOF-ESI-triple quadrupole-ESI-ion trap-ESI-hybrid quadrupole TOF-ESI-ion trap-ESI-orbitrap

A tandem mass spectrometer is a mass spectrometerthat has more than one analyser, in practice usually two.

Tandem mass spectrometry (MS/MS) is used toproduce structural information about a compound byfragmenting specific sample ions inside the massspectrometer and identifying the resulting fragment ions

MALDI TOF spectrum of a peptide mixture

1834.022

1215.597

1969.958

1461.748 2398.323

2335.083

1110.626

952.4722203.004

1269.549

1367.765 2145.0282026.977842.548 1891.0411574.915

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800 1000 1200 1400 1600 1800 2000 2200 2400m/z

1 215.597

1216.5 92

1 217.600

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1214 1215 1216 1217 1218 1219m /z

2 399.312

2 398.323

2400.33 2

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2398 2399 2400 2401 2402m /z

1215.597

1216.592

1217.600

2398.323

2399.312

2400.332

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Monoisotopic mass is calculated using themass of the most abundant naturalisotope of each constituent element

1215.597

1216.592

1217.600

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121 4 1 215 1216 12 17 12 18 1219m /z

1215.597

1216.592

1217.600

12C and 14N

the mass difference between the isotopic peaks is1 amu (1 Da) as isotopes differ in mass by theaddition of 1 neutron which weighs 1 amu

Singly charged ion

Doubly charged ion

MALDI-TOF spectrum of a protein

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739.844

740.347

m/z difference 0.5 Da= doubly charged ion

ESI MS spectrum of a peptide mixture

+20+19

+18

+17

+21+22

+16

+15

+14

+23

+24

+25

p1

p2

-Protein is observed as aseries of different charges-Protein MW can be calculatedbased on adjacent m/z-values

ESI MS spectrum of a protein

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• 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

http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htmhttp://www.ionsource.com/http://planetorbitrap.com/

MS in protein chemistry/proteomics/structural biology

-protein identification-protein MW determination-characterisation of post-translationalmodifications-relative quantification of proteins betweensamples-analysis of protein complexes-essential role in proteomics-MS imaging

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Protein MW determination by MS(NOT= identification)

-for MW determination the protein needs to bein solution without salts and detergents

-usually proteins are first purified withRP chromatography before MW measurement

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

Singly charged ion

Doubly charged ion

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+20+19

+18

+17

+21+22

+16

+15

+14

+23

+24

+25

Protein MW determination, ESI MS

p1

p2

-Protein is observed as aseries of different charges-Protein MW can be calculatedbased on adjacent m/z-values

p=m/zp1=(Mr+z1)/z1p2=[Mr+(z1-1)]/(z1-1)

p= a peak in the mass spectrumm= total mass of an ionz= total chargeMr= average mass of the protein

Protein MW calculation from ESI spectra

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If p1=942.753 and p2=998.067

p=m/zp1=(Mr+z1)/z1p2=[Mr+(z1-1)]/(z1-1)

942.753 =(Mr+z1)/z1942.753z1 = Mr+z1941.753z1 = Mr

998.067 = (941.753z1+z1-1)/z1-1998.067z1-997.067 = 942.753z1-1(998.067-942.753)z1 = 996.06755.314z1= 996.067 => z1=18.0075

Mr = 941.753z1 = 16 951.6 Da

Deconvoluted electrospray mass spectrum of myoglobin

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Protein identification methods:•Peptide mass fingerprinting (PMF)•Identification based on MS/MS data from oneor more peptides

Protein identification bymass spectrometry

•protein of interest is cleaved into peptideswith a specific enzyme•peptides are analyzed by MS (and MS/MS)followed by a database search with theacquired MS (and MS/MS) data

A mass spectrum of the peptide mixture resultingfrom the digestion of a protein by an enzyme,usually measured by MALDI-TOF

Identification based on peptide MW information only

Database search engines create theoretical PMFs forall the proteins in the database and compare these tothe measured PMF from protein X

Peptide mass fingerprint (PMF)

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Protein identification based on MS/MS datafrom one or more peptides

Database search engines create theoreticalpeptide fragmentation patterns for all theproteins in the database and compare theseto the measured MS/MS data

SS

N

C

N

CAlkylation

K

R

KK

K

R

KRTrypsin

intact protein

Reduction

alkylated protein tryptic fragments from the protein

Protein identification by MS

•Protein has to be digested into peptides•Disulphide bridges need to be reduced and alkylatedbefore digestion

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Protein digestion into peptidesbefore MS analysis

Digestion can be done in-solution or in-gelThe enzyme has to be as specific as possibleTrypsin is most commonly used enzyme:

-very specific, quite cheap-cleaves peptide bond after lysines and arginines-tryptic peptides are ’good’ for MS analysisbecause they end up with basic amino acid-works for both in-solution and in-gel digestion

Other enzymes:-LysC, cleaves after lysines-LysN, cleaves before lysines-AspN, cleaves before aspartic acid residues-V8 protease, cleaves peptide bondsexclusively on the carbonyl side of aspartateand glutamate residues-possible to do double-digestions

Chemical cleavage:Cyanogen bromide, cuts after methionines

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In-gel digestion

-works with low-femtomolar amounts of proteins

BUT:-easy to contaminate samples with keratin-the gel staining protocol needs to becompatible with MS-silver-staining: fixing with glutaraldehy cross-linksprotein into gel matrix after which identificationis not possible

Peptide mass fingerprinting

-usually peptides need to be desalted andconcentrated before MALDI analysis-> ZipTips, peptide elution directly onto MALDItarget plate-MALDI matrixincluded in theelution solution

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Peptide mass fingerprintingMALDI, Positive ion reflector mode

-silver-stained spots from 2-DE gels, in-gel digestion+ZipTipping

1590.760

1574.775

1434.642

1606.761

1475.7281450.637

1657.772

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1425 1450 1475 1500 1525 1550 1575 1600 1625 1650m /z

1574.775

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1568 1570 1572 1574 1576 1578 1580 1582m /z

Peak picking parameters important in PMF

Label monoisotopic massesCheck for correct isotope!

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Databases

Publicly available search engines for PMF

•Mascot•ProteinProspector/ MS-Fit•PROWL/ ProFound•Aldente

PMF/ database search

www.matrixscience.com

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-check for known contaminants(these should be removed before search)-possibility to do 2nd pass search

Mass accuracy critical

Usually not manymissed cleavages

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’Normal’ search,max 1missed cleavageallowed

Max 4 missed cleavages allowed

PEPTIDE MASS FINGERPRINTING

• ’quick and easy’• 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• not suitable for complex protein mixtures

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Protein identification based on MS/MS datafrom one or more peptides

-MALDI-TOF/TOF or nanoLC-ESI-MS/MS analysis-suitable for (complex) protein mixtures,especially when combined with LC separationof peptides before MS/MS

Tandem mass spectrometry scan types

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Product ion scanning

C H I L PLH

IIL

Inte

nsity

,cou

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

The ions are first separatedaccording to their m/z ratios

Peptide Fragmentation Nomenclature

Peptides do not fragment sequentially, the fragmentationevents are somewhat random.

The most common peptide fragments observed in lowenergy collisions are a, b and y ions. The b ions appear toextend from the amino terminus (N-terminus), and y ionsappear to extend from the carboxyl terminus (C-terminus).

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Protein identification with MALDI-TOF/TOF

-first, PMF with MALDI-TOF-next, selected precursor ions can be fragmented(TOF/TOF analysis)-MALDI produces singly charged parent ions

product ion spectra not as easy to interpretas in ESI-MS/MS-database search with both PMF and MS/MSinformation

874.168

1060.282340.943

1953.011243.966

479.932111.963

664.960

1825.969

972.188746.0871613.623

1158.227 1448.409

194.917

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250 500 750 1000 1250 1500 1750 2000 2250m /z

1060.282

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MALDI-TOF/TOF fragment ion spectrafrom parent ion m/z 1952

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Sequence Query: One or more peptide mass values associatedwith information such as partial or ambiguous sequence strings,amino acid composition information, MS/MS fragment ion masses, etc.

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Protein identification using nanoLC-MS/MS

-50-75 um i.d. RP-columns, 200 nl/minno need to split the effluent before MS

-DDA= data dependent analysis, can be fullyautomated-suitable for complex protein mixtures,possibility to identify hundreds of proteins inone run

DDA =Data Dependent Acquisition(IDA =Information Dependent Acquisition)

-fully automated experiment, first MS scanfollowed by two or more product ion scans-the acquisition software is set to choosecertain types of ions for fragmentation andto use ’suitable’ collision energy for this ion-in ESI tryptic peptides have usually 2-4 charges

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DDA experiment

Exp 1 = TOF MSExp 2 = Product ion scan from parent IExp 3 = Product ion scan from parent II

TIC = total ion current

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Exp 1 = TOF MS

Exp 2 = Product ion scan I

Exp 3 = Product ion scan II

Search engines for (LC-)MS/MS data

-Mascot, Sequest, OMSSA etc-the programs take the fragment ion spectrum ofa peptide as input and score it against theoreticalfragmentation patterns constructed for peptidesfrom the searched database.

-in practise the user is often limited to usethose search engines which accept thedata format from the mass spec used

-mzXML is a open data format for storage andexchange of mass spec data-raw, proprietary file formats from most vendorscan be converted to the open mzXML format

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Ion scores from individualMS/MS spectra

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-produces huge amounts of raw data-requires efficient data processing tools-different database search programs can producedifferent results from the same raw data-false discovery rate estimation

-peptide level-protein level

Protein identification from complex mixturesusing nanoLC-MS/MS

Protein fragmentation-protein separation by SDS-PAGE/2-DEfollowed by in-gel digestion and MS analysis

Now you’re protein is identifiedWhat else can we find in the data?

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Cytokeratin-18 is cleaved during viral infection,and fragments localize onto mitochondria

Mitochondrial protemes

Based on 2-DE and MS data:N-terminal fragment of theidentified protein

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TAP-tag purification of protein complexes,on-beads digestion with double enzyme,first LysC 2h, then trypsin o/n

Post-translational modifications

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KIN2 phosphopeptide

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-if enough material is available classical Edmandegradation is still a vey good method-partial peptide sequencing is possible basedon MS/MS data

De novo =peptide sequencing performed withoutprior knowledge of the amino acid sequence

De novo sequencing

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Publishing protein ID data

-most biological journals do not have anyguidelines yet

Mat+Met:-MS instrument, identification type-data acquisition+processing software-database search engine

Results: Table from ID results

Mol Cell Proteomicswww.mcponline.org

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In the experimental section:

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http://www.ebi.ac.uk/pride/archive/