BioanalyticsandBiosensors

Spring2011
NielsLion
Niels.lion@mavietonsang.ch

Peptidemassfingerprintingexercise

Inthecourseofahumanplateletproteomicproject,aproteinbandwasexcised
fromaonedimensionalelectrophoresisgelatthetopofthegel(proteinmarkers
indicatethattheproteinislargerthan220'000Da.Theproteinwasexcisedfrom
the gel, reduced with DTT and alkylated with iodoacetamide, digested with
porcine trypsin, peptides were extracted from the gel piece and spooted on a
MALDI plate with CHCA matrix (Cyano4hydroxycinnamic acid). Peptides
wereanalysedonaMALDIQ_TOFinstrumenttoidentifytheprotein.

The mass list obtained from the MS spectrum is appended in the file
Prot_spot.txt.

Firstidentification

1. GothetheAldentewebpage.Copy/pastethemasslistinthepeaklistbox.
2. ChooseUniProt/SwissProtasthedatabase
3. Choosehomosapiensasthetaxon.
4. Extendthesearchmassrangetobesuretoincludetheprotein(higher
than220'000Da.
5. LeavethepIrange014.
6. UntickVarsplices(searchforknownmutations)andFragments(search
forknownproteolyticfragments).
7. Checkthefollowingoptions:Trypsin,1miscleavage,monoisotopic
resolution,[M+H]positiveionmode.UncheckthePTM(posttranslation
modifications).
8. Choosetheappropriatemodificationcorrespondingtoiodoacetamide
alkylation(asfixedmodification).Addmethionineoxidation(MSO)as
variablemodification.LeavetheMaxandScoreparametersunchanged.
9. IntheThresholdsection,increasetheSpectrometershiftmaxto0.5Da,
andtheSpectrometerinternalerrormaxto50ppm.
10. Launchthesearch.

Thereshouldbeonlyonesignificantidentification(scorehighlightedingreen).
Report the identification indicators (score, number of peptide identified,
sequencecoverage,averageshiftinmass,slope)togetherwiththeidentification
parameters.

Secondroundofidentificaiton

Whenyouinspectthedetailsoftheidentificationforthefirstmatch,youseethe
list of matched peptides. Some of them are marked with grey or red asterisks.
Search the help section for the signification of these asterisks and explain in
detailwhattheymean.

Ifyougothothebottomofthefirstidentificationdetails,therearelinkstoother
tools.ClickontheFindPepttoolontherecalibratdspectrumtool.Aldenteisable
to recalibrate the data (compensate for experimental deviations between the
theoreticalandobservedmassesofthepeptides).Brieflyexplainthetheoryof
the recalibration procedure. The Findpept tool allows you to search for
explanation of unidentified peaks in your spectrum (trypsin autoproteolysis,
knownclassicalcontaminants,unspecificcleavages).

OntheFindpeptpage,checkthatcysteinesaremodifiedwithiodoacetamide,tick
the methionine oxidation modification, tick [M+H] positive ion mode, and
monoisotopicresolution.Intheenzymesection,selectporcinetrypsin,andtick
all three boxes (specific cleavage by the enzyme, autolytic cleavage of the
enzyme,specificcleavageofhumankeratins).Launchthesearch.

In the result page of FindPept, you first find the sequence of the protein
identifiedbyAldente,thentheFindpeptsearchparameters,andtheadditionally
identified peaks (first from keratins, then from trypsin autolysis, then peptides
fromtheidentifiedproteincleavedspecificallybytrypsin,thenpeptidesfromthe
identified protein cleaved unspecifically). Manually remove from the original
peaklistthemassescorrespondingtokeratinsandtrypsinautolysis.

Search the internet for the masses of CHCA matrix clusters (the matrix used to
performMALDIionization).Manuallyremovethesemassesfromthepeaklist.

SubmitthecuratedpeaklisttoAldenteagainusingthesameparameters.Report
theidentificationresults(score,sequencecoverage,numberofhits,averageshift
inmass,slope).

Thirdroundofidentification

In the previous round, you have removed all contaminant peaks, which should
haveincreasedtheidentificationscore.Thenyouwilltrytooptimizethesearch
parameters in Aldente: in the threshold tab, modify the spectrometer internal
error (range 10200 ppm) and report the identification results. You should
realizethatthereisalowerlimitbelowwhichthenumberofidentifiedpeptides
diminishes. Do this lower limit corresponds to the known mass accuracy of a
MALDIQTOF instrument (provide background information for this
parameter)?

You have now defined an optimal mass accuracy (spectrometer internal error
max) that corresponds to the mass accuracy of the mass spectrometer in this

experiment that is a compromise between search stringency and realistic MS

accuracy.

Fourthroundofidentification

Usingtheoptimalspectrometerinternalmaxerror,gotothepeptidetabandtick
thePTMboxtoallowthesearchforposttranslationmodificationsinyourpeak
list. Launch the search, and report the additional peptide identifications with
posttranslationalmodifications.

ByclickingontheproteinaccessionnumberintheAldenteresultpage,youwill
access the Uniprot protein database summarising all known information about
theidentifiedprotein.CheckiftheadditionalidentificationofPTMscorrespond
toknownPTMs.Reportthefinalidentificationresults(score,sequencecoverage,
numberofpeptideidentified,identifiedPTMs.)