MASCOT

ProteomicsSession 11Analysis mass spectrum andprotein annotation (MASCOT)Useful proteomic resourcehttp://tw.expasy.org/

Useful proteomic resourceMatrix Science - Mascothttp://www.matrixscience.com/

Three major functions in Mascot• Peptide Mass Fingerprint (PMF): The experimental dataare a list of peptide mass values from an enzymatic digest of a protein.(MALDI-TOF)• Sequence Query: One or more peptide mass valuesassociated with information such as partial or ambiguous sequencestrings, amino acid composition information, MS/MS fragment ionmasses, etc. A super-set of a sequence tag query.• MS/MS Ion Search: Identification based on raw MS/MS datafrom one or more peptides. (LC/MS/MS)Difference between MALDI-TOF and LC/MS/MSMALDI-TOFLC/MS/MS

PMF analysisRaw data for PMFm/zRelative intensity899.2076 2980.8123905.2126 1471.3723909.1917 2312.2317915.2181 1533.8486925.4637 1881.7635938.3972 1528.94621044.3007 2111.94821050.3141 2396.15501060.2797 4689.06981066.2889 7302.00291072.2991 5688.85111078.3169 8919.11131084.2657 1474.59001088.2793 3180.51221094.2947 4573.51951104.2638 1546.46521110.2837 1470.97341271.3163 1498.0187

Mascot PMF query formMascot PMF parameters• Your name; Email• Search title• Database• Taxonomy• Enzyme• Monoisotopic or Average• Modifications• Protein Mass• Peptide tol. ±• Mass values• Missed cleavages• Data file• Query

DatabaseDatabaseCommentESTMSDBNCBInrSwissProtEST divisions of Genbank,(currently EST_human, EST_mouse, EST_others)Comprehensive, non-identical protein databaseComprehensive, non-identical protein databaseHigh quality, curated protein databaseTaxonomy• Ensure the hit list will only contain entries fromthe selected species1. bring a weak match2. speed up a search

EnzymeNameCleaveDon't cleaveN or C termTrypsinKRPCTERMArg-CRPCTERMAsp-NBDNTERMAsp-N_ambicDENTERMChymotrypsinFYWLPCTERMCNBrMCTERMFormic_acidDCTERMLys-CKPCTERMLys-C/PKCTERMPepsinAFLCTERMTryp-CNBrKRMPCTERMTrypChymoFYWLKRPCTERMTrypsin/PKRCTERMV8-DEBDEZPCTERMV8-EEZPCTERMCNBr+TrypsinMKRPCTERMCTERMNonesee notessemiTrypsinsee notesMonoisotopic or Average MS• nominal mass values: calculated from integer atomic weights. (H=1, C=12,N=14, O=16), not practical in proteomics.• Average mass values: equivalent to taking the centroid of the completeisotopic envelope• Monoisotopic mass value: the mass of the first peak of the isotope distribution.

Monoisotopic or Average massFor peptides and proteins, the difference between an average and amonoisotopic weight is approximately 0.06%.Insulin (5.8 kD)Albumin (66.4 kD)MonoisotopicTol: 1 Da-1.01Monoisotopic MW

AverageTol: 1 DaAverage MWMonoisotopicTol: 2 Da

AverageTol: 2 DaModifications• Most protein samples exhibit some degree of modification.• Natural post-translational modifications: phosphorylation andglycosylation.• Deliberate modifications: deliberately introduced during samplework-up, such as cysteine derivatisation.

ModificationsFixed modifications are applied universally, to every instance of thespecified residue(s) or terminus.Example: Carboxymethyl (Cys) means that all calculations will use 161 Da asthe mass of cysteine.Variable modifications are those which may or may not be present.Example: if Oxidation (Met) is selected, and a peptide contains 3 methionines,Mascot will test for a match with the experimental data for that peptidecontaining 0, 1, 2, or 3 oxidised methionine residues.Peptide tol. ±The error window on experimental peptide mass values%mmuppmDafraction expressed as a percentageabsolute milli-mass units, i.e. units of .001 Dafraction expressed as parts per millionabsolute units of Da

Missed cleavagesMissed cleavage = 0, complete digestionMissed cleavage >=1, incomplete digestionSubmit and processing

Concise protein summaryprotein summary

PMF protein view (I)Protein nameScore and ExpectMW and pIcoverageLarger proteins tend to come with a lower sequence coveragePMF protein view (II)Match peptidesRMS errorNo match peptidesProtein information

Possible counting algorithm of PMF in MASCOT• Higher score will show if1. More matched peptidesGenerally with more than 5 matched peptides will geta significant match2. Lower tolerance allowed3. Fewer missed cleavage allowed4. Fewer unmatched input MS signals (trash signal)• Lower score will show if1. Fewer matched peptidesGenerally with less than 4 matched peptides will getan insignificant match2. Higher tolerance allowed3. More missed cleavage allowed4. More unmatched input MS signals (trash signal)5. Consideration of “variable modification”MS/MS analysis

Raw data for MS/MSParent ionDaughter ionMascot MS/MS query form

Protein summaryMost possible candidateMS/MS Protein view (I)The sum of all highest scores within each peptide group

MS/MS Protein view (II)Protein score: The sum of all highest scores within each peptide groupPeptide view

Matching algorithm of MS/MS ion searchMatched to KQSLGELIGTLNAAK (a 15 amino acid peptide)b1-y14b2-y13…………….K, KQKKQSAK NAAKAKKQSLGTLNAAKNAAKIGTLNAAKKQSLGELKQSLLNAAKKQSLGGTLNAAKKQSLGELIGTLNAAKKQSLGELILIGTLNAAKKQSLGELIGTELIGTLNAAKGELIGTLNAAKTo obtain a significant match, not all b and y ions of a peptide have to be found