Mascot: an [email protected]
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 Step
2
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
7
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
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 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!
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