CS188-MassSpectrometry

8/9/2019 CS188-MassSpectrometry

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An Introduction to Mass Spectrometry

by

John J. Kelley, III


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Overview of MassSpectrometry

Mass Spectrum

Mass Analyzer

IonizationM+/FragmentationSample

Molecule M!


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Mechanisms of Sample

Ionization"rotonation# M + $ + M$ +

%ationization# M + %at + M%at +

&eprotonation# M$ M ' + $ +

(lectron ()ection# M M + . + e'(lectron %apture# M + e' M '.


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The Components of aMass Spectrometer

Inlet system

Ion Source Analyzer Ion

Detector

Computer Mass Spectrum

m/z


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Types of

SpectrometersMagnetic Sector Instruments*uadrupole Instruments

ime'o 'Flight InstrumentsIon rapFourier rans orm Ion %yclotron-esonance F 'I%-!


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Magnetic Sector MassSpectrometer


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Quadrupole Mass Spec


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Time of Flight Mass Spec


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Time-of-Flight MassSpectrometry

here are actually t o type o mass

analyzers in use or MA &I#

0 -e lectron time'o ' light mass spectrometer

0 inear time'o ' light mass spectrometer


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$ecay

inear 1F machines' ragmented in light by only

mass. (nsemble o decay products tra2el ithparent ion.I , ho e2er, a retarding ield is placed in ront o thedetector, the e3tent o ragmentation is easilymeasured

he measuring principle uses the act that the4inetic energy o a ragment ion is proportional to its

ractional mass. hus, in the retarding ield o there lectron, such ragments ill turn around earlierthan their respecti2e precursors and ill arri2e at thedetector sooner.


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#ost Source $ecay %#S$&fragment ion mass analysis

e2aluation o post source decay ollo ing MA &I-. Kau mann, J. Biotechnol. 4, 567 5889!.


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#ost Source $ecayhus, it came rather as a surprise that roughly

the same 4ind o in ormation,as in the :proteinladder; se


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'pplication of Mass Specto (ioMolecules

(SI'MS# (lectrospray Ionization MassSpectrometry

MA &I'MS# Matri3 Assisted aser&esorption Ionization Mass Spectrometry


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>loc4 diagram o MA &I' inear 1F MS

&igital 1scilloscopec o m pu t e r

Cotter, R.J. In Time-of- li!"t #ass Spectrometry$ Cotter, R.J., %d.$ American C"emicalSociety& 'as"in!ton, DC, ())4$ pp. 4(.


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Finnigan Corporation+s (enchtop)'S, M'T ...

*peration of t"e +AS%R#AT is strai!"tfor ard, it" no need for instrument tunin! or

optimization. Samples ran!in! from small peptides up to lar!e !lycoproteins o/er ,Da can 0e easily and rapidly measured.

f l h


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In time'o ' light 1F! instruments, positi2eions are produced periodically bybombardment o the sample ith briepulses o electrons, secondary ions, or inthis case laser'generated photons.

hese laser pulses typically ha2e are


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he ions produced by the laser are thenaccelerated by an electric ield pulse o 57 @ to 57 A B that has the same re


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Time-of-Flight Mass Spectrometry

All ions entering the tube ideally ha2e the same4inetic energies, their 2elocities in the tube must2ary in2ersely ith their masses, ith lighterparticles arri2ing at the detector earlier than thehea2ier ones.

he ions then dri t through a ield' ree path,usually 5'@m in length, and are separated inspace and time'o ' light according to the e


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Mechanisms of matri/ assisted laserdesorption0ionization

he matri3 ser2es three ma)or unctions # 0 Isolation o the biomolecules rom each other

T"e 0iomolecules are incorporated in a lar!ee1cess of matri1 molecules, stron!

intermolecular forces are t"ere0y reduced 2matri1isolation3.*ne of t"e most important conditions formatrices to or ell is an intimate mi1in! of t"e

analyte and t"e matri1.optimal molar ratios are in t"e (&( to (&(2analyte to matri13 ran!e. T"is means t"at analytemolecules are sin!ularly and fully surrounded

2sol/ated3 0y matri1 molecules.


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The principle of matri/assistance

0 Absorption o energy rom the laser light

he matri3 molecules absorb the energyrom the laser light and trans er it into

e3citation energy o the solid system.

0 hereby inducing an instantaneous phase

transition o a small molecular layers o thesample into a gaseous species.

T /


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T e pr nc p e o matr /assistance

0 Ionization o the biomoleculesAn acti/e role of t"e matri1 in t"e ionization oft"e analyte molecules 0y p"otoe1citation orp"otoionization of matri1 molecules, follo ed0y proton transfer to t"e analyte molecules isli ely, t"ou!" not pro/en une5ui/ocally todate.In practice, success or failure of a matri1depends on properties ot"er t"an actin! as anener!y transfer mediator 6nfortunately, none of t"e many t"eories sofar proposed can fully descri0e t"e c"ain of

e/ents.


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The most commonly usedsu1stances

Matri3 Ca2elength %omments?,9'&ihydro3y benzoic acid &$>! @@D

&$> + 57E 9'metho3y salicylic acid @@D used for masses 7 Da

=icotinic acid ?66 nm

'$ydro3y picolinic acid @@D used for oli!onucleotides

lycerol ?.8 m li5uid matri1

G. >ahr, M. Karas, F. $illen4amp, Fresenius J. Anal. Chem. 849 , DH 588 !.


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Sample #reparation

Simply mi3 57 nanomoles o an a


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2ses of M')$I-TOF in (iomolecule'nalysis

"rotein Analysis 0 Mass determination o >iomolecules

#a1imum size of protein t"at can 0e measured,

:uman Immuno!lo0ulinSin!le Cell analysis of neuropeptidesad/anta!es o/er classical met"ods of proteinanalysis

0 "rotein adder Se


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Size Determination of :uman immuno!lo0ulin I!#0y #A+DI , #' ;


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(asic Structure of a 3euroncell of the nervous system specialized to generate

and transmit nerve impulses

soma A3on

A3on terminal

commissure

=entral

dorsal

P tid d b th N


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Peptides used by the NervousSystem

"eptides are idely used by the ner2oussystem as neurohormones to regulate di erentprocesses such as reproduction, gro th,metabolism and beha2ior.

he peptides are synthesized in the orm olarger prohormones, rom hich they areproteolytically clea2ed, urther modi ied, and

stored in secretory granules in a3on terminals,rom hich they are released in response to

depolarization


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sin!le dorsal CDC cell


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intercere0ralcommisure of adifferent animal

sin!le dorsal CDC cell

sin!le /entral CDC cell

intercere0ral commisure

%.-. Jimenez et al., J. Neurochem. > , 76


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M')$I-MS vs Classical ScreeningMethods for mass determination

In many instances, MA &I'MS can gi2ein ormation o anted or un anted targets rightout o the ra or at least crude material

arget protein identi ication using MA &I'MS2s S&S'"A ( 0 SDS-PA?% Sodium Dodecyl sulfonate

polyacrylamide !el electrop"oresis 0 T"e .(@- . (@ accuracy of molecular ei!"t

determination /s -( @ it" SDS-PA?%


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M')$I-MS vs Classical ScreeningMethods

Short measuring time and negligiblesample consumption 0 e minutes and subpicomolar amounts

Cind all pro its such as in ormation onmicroheterogeneities, glycosylation,phosphorylation


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Classical #eptideSe4uencing

he most direct se


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,dman $egradation

A multi'step process# 0 A chemical reaction is used to remo2e

one amino acid at a time rom the aminoterminal

0 (ach amino acid is con2erted to a stableorm and then identi ied by analytical

re2erse'phase high'per ormance li


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Gsing MA &I' 1F toSe


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A Step ise &egradation

%arried out ith a small amount oterminating agent present in the couplingstep 0 A mi3ture o p henyl iso thio c yanate "I %! plus

9E 2/2 p henly iso c yanate "I%! is used as theterminating reagent.

"henylisocyanate reacts ith the =$ ?

group o a polpeptide chain to yield an = 'phenylcarbamyl'peptide, hich is stable tothe conditions o degradation


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5ydrolysis Stephe trif luoro a cetic acid FA! is used to

clea2e the terminal a mino a cid AA! rom thep henyl thio c arbamyl " %! peptide.

he p henyl c arbamyl "%! peptides ormedare stable to the tri luoroacetic acid used toclea2e the terminal amino acid rom thephenylthiocarbamyl " %! peptide.

' S i d d i


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' Stepwise degradation

PC- AA -AA 8-AA 4-AA - -AA n PC-AA ( -AA -AA 8-AA 4-AA - -AA n

PC-AA 8-AA 4-AA - -AA n one'step MA &I'MSladderse. %hait et al., Science > , H8 588@!.


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One step M')$I-MS readout

m/z

N CH

H

H C

O

H

CH2

CH 2

C

O OH

"% AA? AAn

m

PC- AA ( -AA -AA 8-AA 4-AA - -AA n PC- AA -AA 8-AA 4-AA - -AA n

@1 H

9 % 67

5 = 5

H $ H

LLLLLLLL

m 5?8.D


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#rotein ladder Se4uencing of 67lu 89:1rinopeptide (

>. %hait et al., Science > , 87 588@!.

' d ill i


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' second illustration"rotein adder se


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$ gel of erythroleu=emia cell


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$-gel of erythroleu=emia cell


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7oal of the future

the 4ey is to identi y a spot on a ?&'gel

earn something about it that can beused to search protein databases

Identifying proteins from two


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Identifying proteins from two-dimensional gels

0 1ne step to ard this goal

as sol2ed by se2eral groups in the 58H7sho de2eloped methods to trans er spotsrom gels onto membranes so they could be

analyzed

0 &ra bac4Identi ication o all the resol2able proteins ona t o'dimensional gel by con/entional

protein se


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#utting it all together* Identifyingproteins from $-7els

"roteins are electroblotted rom t o'dimensional gelshe spots are e3cised and digested ith trypsin

0 rypsin clea2es the %'terminal end o arginine and

lysine producing peptide ragmentsMasses are determined at the subpicomole le2el byMA &I'MS o the tryptic digest

F-A FI , a computer program, then searches theprotein se


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he protein database used

%onsist o a combination o se2eral idely a2ailabledatabases# S iss-Prot , Protein IdentificationResource , and a translation o ?enEan

he resulting database contains 5H.H millionresidues, representing o2er 85,777 entries

he %'language source code or F-A FI can beobtained 2ia ('mail to c4 Ngene.com


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From 7enome to #roteome

It sounds li4e mission impossible# 0 Follo the changes ta4ing place inside a cell byidenti ying the thousands o di erent proteinsthe cell produces and atching ho theychange and lo o2er time.

0 o a gro ing band o researchers, ho e2er,such a mission is becoming more realistic,

than4s to the increasing 2olume o se


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M )$I TOF Spectra

T l f h F


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Tool of the FutureMA &I'MS coupled ith this method

acilitates simultaneous processing o alarge number o t o'dimensional gel spotsresearchers are e3cited about theimplications# 0 &rugs induce changes in protein patterns

these changes on the gels should gi2e important

clues about both the drugs desirable e ects and itsto3icity

0 Knoc4 out their a2orite gene and see its e ecton the protein pattern

CS188-MassSpectrometry

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