Mascot: an introductionblent@gel.ym.edu.tw

Basicso What is Mascot?

o It is a search engine which uses mass spectrometry (MS) data to identify proteins from primary sequence databases

o What Mascot offers in proteomics study?

tool input output

Peptide Mass Fingerprint

peptide mass values

Protein hits

Sequence Querypeptide massamino acid seq, etc.

MS/MS Ion SearchPeak lists of mass-intensity pairs

Peptide Mass Fingerprint brief

o Rationaleo Due 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

o Disadvantageo However, it only can identify protein with

known sequence

Peptide Mass Fingerprint Search

Peptide Mass Fingerprint Step

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return the result via e-mail, save ur time!

Peptide Mass Fingerprint Step

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All Sequence DB Mascot supported

Why there is no selection option ‘EST’ DB in peptide mass fingerprint?

Peptide Mass Fingerprint Step 3

Special taxonomy id1. unclassified (undefined or unmatched, 1.5k in NCBInr)2. species info. unavailable

limit ur search, save ur time!

Peptide Mass Fingerprint Step

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Peptide Mass Fingerprint Step

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protein modification

Peptide Mass Fingerprint Step

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protein size peptide size tolerance

Peptide Mass Fingerprint Step

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In mass calculation, regard molecule is1. MH+ (positive charged)2. Mr (neutral, no charge)3. M-H- (negative charged)

choice of m/z value

Peptide Mass Fingerprint Step

8Mascot generic file format

example 1: peptide only mass

example 2: peptide mass and intensity

example 3: with embedded parameters

Example Result

Exerciseo give 2 sets of peptide mass values,

please use peptide mass fingerprint to find the proteins possible composed by these peptides

o hints are included in data file

Sequence Query new options

Sequence Query file syntax

o expressiono m [seq()] [comp()] [ions()] [tag()] [etag()]o m: mass

o new information to limit the searcho seq (orientation prefix + regular expression)

o orientation prefix: b, y, *, n, co allow multiple seq() in one line (query)

o comp (#1[aa1]#2[aa2]#3[aa3]…)o ions ([b/y]-m1:i1,m2:i2, m3:i3…)o tag and etag

o expression (first peak mass, inferred a.a., last peak mass)o search sequence bidirectionally o some modification will change the mass of peptide (set it in

etag)o more other options!

Sequence Query example

from experimental paper study

MS/MS ion search new options

one can assign mass here(some file formats do not have mass record

Mass spectrometer 3 main parts

o ionizer (ionize molecule into ions) o MALDI (Matrix-assisted laser desorption ionization)o ESI (ElectroSpray ionization)

o mass analyzer (measure the ions)o F = q(E+v×B) = ma, m/q = (E+v×B)/a, noted as m/zo sector MSo TOF (time of flight) MSo Q (quadrupole) MS, QIT (quadrupole ion trap) MSo FT (Fourier transform) MS

o detectoro m/z (mass-to-charge ratio)o record the relative intensities of molecules with different

m/z