Overview of Mass SpectrometryMass SpectrumMass AnalyzerIonization M+/FragmentationSample Molecule (M)Protonation : M + H+MHCationization : M + Cat+MCat+Deprotonation: MH M-+ H+Electron Ejection: M M+.+ e-Electron Capture: M + e- M-.Mechanism of Ionization
1. Sample ismixed inmatrix anddried ontarget.2.Target is introducedinto high vacuum ofMS.3.Sample is irradiatedwith laser desorbingions into the gas phaseand the clockmeasuring the time offlight starts.4.Ions are accelerated by an electricfield to the same kinetic energy andthey drift down the field free flighttube where they are separated inspace5.Ions strike thedetector at differenttimes depending onthe mass to chargeratio of the ions6.A data system controls allthe parameters, acquires thesignal vs. time and permitsdata processingSchematic of a MALDI-TOF Experiment
Schematic representation of a triple quadrupole MSinstrumentQuadrupole mass analyzer Trajectory of an ionOperation in full scanmodeOperation in MS/MSmode
CID CID is divided into low energy (<100 eV) and highenergy(>1000 eV) based on the collision energy. High energy CID produces more fragments, but is morecomplicated to interpret. Low energy CID has a limit on the m/z it can dissociate ofapproximately 1000.
Interpretation of Tandem Mass Spectra ofPeptides Known Sequence- Calculate expected fragments andcompare to tandem spectra to see match Modified Sequence-Calculate unmodified sequence compareto tandem spectra to see difference where modificationoccurs. Unknown Sequence- Check Database to see if it is a match Unknown Sequence not in Database- Manual Interpretation(Practice!Practice!Practice!)
Manual Interpretation Goal-Assign as many abundant fragments as possible toa spectra Remember Cysteine modifications Know type of fragments that are typically observed bydissociation method. Low Energy (b and y, loss of neutrals from thesefragments) High Energy (x,y,z, a,b,c, v, d,w)
MS/MS: Tandem Mass SpectrometryLaser, 200Hz Choice of DifferentCollision Gases(He, Ne, Ar, Kr, Xe)High CE
Average residue masses of amino acidsThe side chains that give amino acid its special chemistry are attached tothe alpha carbon.To complete the structure add an extra proton( 1amu) to the N-terminalresidue and an extra OH (17 amu) to the C- terminal amino acid.
MS/MS Spectrum Representation For peptide IYEVEGMRDistance between peaks on m/z axis is used to determinepartial sequence of the peptide
Possible b- and y-ion fragments for the peptideAVAGCAGARThe b- ion series complements the y-ion series.The gap between the b₇ and b₆ ions is 57, which correspond to glycine.The gap between b₆ and b₅₅ is 71, which corresponds to alanine.The complement b-ion series(b₈ through b₁) corresponds to theAVAGCAGA motif.Thus he y- ion and b-ion series describes the same amino acid sequec intwo different directions.
Structures of the b₄ and y₅ ions from cleavagebetween the glycine and cysteine residuesB₄ m/z 299.305 Y₅ m/z 476.557
Annotated MS-MS spectrum of the [M+2H]²⁺ ion ofAVAGCAGAR showing b- and y- ions
Digest protein mixturesPeptide ionsExperimental MS/MS SpectraSearch databases for peptidecandidates with similar precursormassTheoretical spectra generatedScore candidate peptideValidate peptideHighest score: PeptideidentificationValidate protein
Charged Parent ionFragmentation of the amicle backboneA, a (no H+ added) X, x (no H+ added)
Charged Parent ionB, b (no H+ added) Y”, y+2 ion or y ion, 2H+ addedGroups formed at cleavage point
Charged parent ionb₂ ionImmonium ionImmonium ionsThe low mass region of MS/MS spectra often containions that are indicative of the presence of specific aminoacids in the peptides.These immonium ions arise from at least two internalbond cleavages.Labeled with the single letter code for the parentamino acid.
PRTEINPRTEYNPWTEYNb₁b₁b₁y₁y₁y₁b₂b₂b₂y₃y₃y₃y₂y₂y₂b₃b₃b₃b₄b₄b₄y₄y₄y₄b₅b₅b₅y₅y₅y₅100 200 500400300 700600Theoretical spectral of peptidesPRTEIN and PRTEYN(one mutation) PWTEYN (two mutation)Representing masses of all the b and y ion in the corresponding peptides.
Mechanism of C-terminal cleavage after Asp residueThe carboxylic acid group of Aspis often involved inrearrangement reactions.In the gas phase group canattack the amide bond giving riseto a cyclic intermediate thatbreaks down to give a b-ionwhich often dominates theMS/MS spectrum.No C-terminal cleavage is foundfor Pro
Loss of C-Terminal amino acid When the C-terminal amino acid residue is hydrophobic(mostcommonly Phe and Tyr and to a lesser extent Leu, Ile, Val) an ioncorresponding to the loss of residue mass is observed in additionto the y-ionThis is due to intramolecular rearrangement.
Low energy production of a-ionsCharged b ionzCharged a ionzMechanism of a ion formationMost of the a ion observed in lowenergy MS/MS spectra are notformed by direct cleavage of thepeptide bond as observed in highenergy spectra.They are formed by loss of COgiving rise to the formation of a-and b- ion doublets separated by 28mass units.
Loss of 18,34 and 105 mass unitsLoss of water occur very often from serine and threonineresidue.Cysteine show loss of 34 due to H₂S.Cysteine is deliberately modified to increase the digestionefficiency and prevent digetion bond formation.The most common modifications, with vinylpyridine to formpyridylethylscysteine or idoacetic(or idoacetamide) to givecarboxymethylcysteine, give rise to losses of 105 and 92respectively.Another common loss seen is 48 from metheonine containingpeptides, this is the loss of methylsulphide.
Immonium ionsAmino acids Immonium ions Related ions CommentsAlaArgAspAsnCysGlyGlnGluHisIle/LeuLysMetPheProSerThrTrpTryVal4412988877630101102110861011041207060741591367270,87,10081846191130107Marginally usefulm/z 129 weakother(4:2:1)Often weak or absentOften weakRelatively weakNot usefulm/z 101 often weakVery strongStrong110:81(3:1)Strong 86:84 (4:1)Usually weak or absentStrong 104:61 (3:4)Strong 120:91 (3:1)Strong 130:159 (1:2)Strong 136:107 (1:2)Fairly strong
Relative amino acids mass and their behavior in MS/MSRESIDUE MASS POSSIBLE EQUIVALENT MASS BEHAVIOUR IN MS/MSGly 57 Give weak signalAla 71 -Ser 87 loses -18( water )Pro 97 strong signal after C-terminal cleavagegiving internal ions.Val 99 AcGly (N terminal only)Thr 101 loses -18( water)Cys 103 unusual, alwaysmodified ,lose -34(H2S)Pyro-Glu 111 N-terminal onlyLeu/Ile 113 AcAla (N-terminal only) -Asn 114 Gly-Gly loses -17(ammonia)
RESIDUE MASS POSSIBLE EQUIVALENT MASS BEHAVIOUR IN MS/MSAsp 115 Gly-Ala Cleave N-terminal to givestrong signals.Lys 128.09 Gly-AlaGln 128.06 Strong lose of -17Glu 129 AcSer( N- terminal only) -Met 131 lose of CH₃SH -48His 137 like Pro but weaker .lookfor 110.Phe 147 Can be Met(Ox)Arg 156 Gly-Val/AcAsn (N-terminal only)CmCys 161 -Tyr 163 No lose of 18Trp 186 Gly-Glu or Ala-Asp or Ser-Val Like Pro and His but muchweaker internals.
Unknown spectrumDatabase searchDe novoidentificationCompare to model spectra ofcomplete peptidesPartial sequence
Number of peptide candidatesUsing enzyme cleavage specificities canreduce the number of candidates byapproximately one order of magnitude.Inclusion of additional post-translational modification can increasethe number of candidates.
Manual Interpretation GuideAccurate mass detection of native parent, methylated and acetylated ions.Depending on parent charge deconvolute spectrum to all 1+ ions. Remove allspikes.Print out spectrum as is , and then with all regions scaled up to the same size.Label all possible a-b pairs (a=b-28).Label -18 an -17 losses for water and ammonia respectively.Examine low mass end.Look for PHW immonium ions.If PHW present, look for intense internal cleavage series.Inspect High mass end for b and y ions and residue mass loss.Look for ion series for by sequentially subtracting residue masses.Look for corresponding b/y ion pairs.For each residue look for diagnostic losses from it and respective immoniumions.Be careful about the dipeptide masses that match residue masses.Assign tentative sequence taking into account acetylation data.Compare all theoretical masses to data. Are all ions accounted for? If notrepeat the process.