Mascot: an introduction

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Mascot an introduction

• blent_at_gel.ym.edu.tw

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Basics

• What is Mascot?
• It is a search engine which uses mass
  spectrometry (MS) data to identify proteins from
  primary sequence databases
• What Mascot offers in proteomics study?

tool input output
Peptide Mass Fingerprint peptide mass values Protein hits
Sequence Query peptide mass amino acid seq, etc. Protein hits
MS/MS Ion Search Peak lists of mass-intensity pairs Protein hits
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Peptide Mass Fingerprint brief

• Rationale
• 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
• Disadvantage
• However, it only can identify protein with known
  sequence

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Peptide Mass Fingerprint Search
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Peptide Mass Fingerprint Step 1
return the result via e-mail, save ur time!
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Peptide Mass Fingerprint Step 2
Why there is no selection option EST DB in
peptide mass fingerprint?
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Peptide Mass Fingerprint Step 3

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

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Peptide Mass Fingerprint Step 4
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Peptide Mass Fingerprint Step 5
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Peptide Mass Fingerprint Step 6
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Peptide Mass Fingerprint Step 7
choice of m/z value

• In mass calculation, regard molecule is
• MH (positive charged)
• Mr (neutral, no charge)
• M-H- (negative charged)

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Peptide Mass Fingerprint Step 8
example 1 peptide only mass
Mascot generic file format
example 2 peptide mass and intensity
example 3 with embedded parameters
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Example Result
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Exercise

• give 2 sets of peptide mass values, please use
  peptide mass fingerprint to find the proteins
  possible composed by these peptides
• hints are included in data file

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Sequence Query new options
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Sequence Query file syntax

• expression
• m seq() comp() ions() tag() etag()
• m mass
• new information to limit the search
• seq (orientation prefix regular expression)
• orientation prefix b, y, , n, c
• allow multiple seq() in one line (query)
• comp (1aa12aa23aa3)
• ions (b/y-m1i1,m2i2, m3i3)
• tag and etag
• expression (first peak mass, inferred a.a., last
  peak mass)
• search sequence bidirectionally
• some modification will change the mass of peptide
  (set it in etag)
• more other options!

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Sequence Query example
from experimental paper study
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MS/MS ion search new options
one can assign mass here (some file formats do
not have mass record
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Mass spectrometer 3 main parts

• ionizer (ionize molecule into ions)
• MALDI (Matrix-assisted laser desorption
  ionization)
• ESI (ElectroSpray ionization)
• mass analyzer (measure the ions)
• F q(EvB) ma, m/q (EvB)/a, noted as m/z
• sector MS
• TOF (time of flight) MS
• Q (quadrupole) MS, QIT (quadrupole ion trap) MS
• FT (Fourier transform) MS
• detector
• m/z (mass-to-charge ratio)
• record the relative intensities of molecules with
  different m/z