1. Mascot: an introduction blent@gel.ym.edu.tw

2. Basics oWhat is Mascot? oIt is a search engine which uses mass spectrometry (MS) data to identify proteins from primary sequence databases oWhat Mascot offers in proteomics study? toolinputoutput Peptide Mass Fingerprintpeptide mass values Protein hits Sequence Query peptide mass amino acid seq, etc. MS/MS Ion SearchPeak lists of mass-intensity pairs

3. Peptide Mass Fingerprint brief oRationale oDue to the specificity of peptide mixture resulting from the digestion of a protein by enzyme, it becomes possible to identify protein from MS data of peptides alone oDisadvantage oHowever, it only can identify protein with known sequence

4. Peptide Mass Fingerprint Search

5. Peptide Mass Fingerprint Step 1 return the result via e-mail, save ur time!

6. Peptide Mass Fingerprint Step 2 All Sequence DB Mascot supported Why there is no selection option ‘EST’ DB in peptide mass fingerprint?

7. Peptide Mass Fingerprint Step 3 Special taxonomy id 1.unclassified (undefined or unmatched, 1.5k in NCBInr) 2.species info. unavailable limit ur search, save ur time!

8. Peptide Mass Fingerprint Step 4

9. Peptide Mass Fingerprint Step 5 protein modification

10. Peptide Mass Fingerprint Step 6 protein sizepeptide size tolerance

11. Peptide Mass Fingerprint Step 7 In mass calculation, regard molecule is 1.MH + (positive charged) 2.Mr (neutral, no charge) 3.M-H - (negative charged) choice of m/z value

12. Peptide Mass Fingerprint Step 8 Mascot generic file format example 1: peptide only mass example 2: peptide mass and intensity example 3: with embedded parameters

13. Example Result

14. Exercise ogive 2 sets of peptide mass values, please use peptide mass fingerprint to find the proteins possible composed by these peptides ohints are included in data file

15. Sequence Query new options

16. Sequence Query file syntax oexpression om [seq()] [comp()] [ions()] [tag()] [etag()] om: mass onew information to limit the search oseq (orientation prefix + regular expression) oorientation prefix: b, y, *, n, c oallow multiple seq() in one line (query) ocomp (#1[aa1]#2[aa2]#3[aa3]…) oions ([b/y]-m 1 :i 1,m 2 :i 2, m 3 :i 3 …) otag and etag oexpression (first peak mass, inferred a.a., last peak mass) osearch sequence bidirectionally osome modification will change the mass of peptide (set it in etag) omore other options!

17. Sequence Query example from experimental paper study

18. MS/MS ion search new options one can assign mass here (some file formats do not have mass record

19. Mass spectrometer 3 main parts oionizer (ionize molecule into ions) oMALDI (Matrix-assisted laser desorption ionization) oESI (ElectroSpray ionization) omass analyzer (measure the ions) o F = q( E +v× B ) = ma, m/q = ( E +v× B )/a, noted as m/z osector MS oTOF (time of flight) MS oQ (quadrupole) MS, QIT (quadrupole ion trap) MS oFT (Fourier transform) MS odetector om/z (mass-to-charge ratio) orecord the relative intensities of molecules with different m/z