Mass Fingerprint
Dec 18, 2015
Mass Fingerprint
Protease
• A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain.
• Or: a protease breaks protein in water.• Trypsin is one protease that is commonly used in mass spec
analysis of proteins.
…M-A-L-R-Q-V-…
…M-A-L-R Q-V-…
R- G- F - K- I - A - E - W - M
MW (Average mass):1136
Trypsin Trypsin
Treatment with trypsin gives 3 different fragments: 1. R (MW 174) 2. Gly - Phe – Lys (MW = 57+147+128 + 18 = 350) 3. Ile-Ala-Glu-Trp-Met (MW = 113+71+129 + 186 +18 = 648
X-X-X-X-X-X-X-X-Rn-1 Rn R n+1
Trypsin cleaves at only at peptide bond Rn-1 = K, R; Rn P
Note: each internal peptide will end with Lys (K) or Arg (R)
Example:
Cleavage with Trypsin (tryptic digestion)
Mass fingerprint
• 1. Cleave the protein at certain sites (get peptides)
• 2. Measure the masses of the peptides.• 3. Find a protein in the database with the
same theoretical peptide masses.
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000m/z0
100
%
2790.22
1324.60
1265.62
1179.41
2789.22
1325.62
2466.18
2465.20
1759.931326.60
1477.62
1327.611460.59
1748.86
1478.611540.63
1974.94
1760.93
1761.92
1975.93
2356.102355.111976.92
2179.87
2467.19
2468.20
2469.172746.23
2791.23
2792.23
2793.23
3104.412794.20
3103.432795.06 3106.42
internal calibrants
Mass “Fingerprint” of a Pure Protein
Peptides from trypsin self-digestion
MALDI-TOF/R MS of Peptides from a Tryptic Digest
Search a database for match
RPSESSYKVHRYAKSGGS another protein ……
in-silicon digestion
in-silicon digestion
……
……
Score Method (Naïve)
• Count the number of matched peaks – allowing a small mass tolerance when matching
• Problem:– Different peaks have different intensity– Some peaks have more proteins match than some
other peaks
Mass Accuracy is Important
Score Method (Better)
• At each mass window, count how often a protein contains a peptide in it.
• Each peak contributes a score log(1/f), where f is the frequency a protein contains a peptide matching the peak.
• Add up the scores of peaks.
Mascot interface
1. Cut spots from 2D Gel, destained and tryptic digest each spot ( Medium to high silver stained spot)
2. Extract peptides and purify by ZipTip
3. Mix with matrix and analyze by MALDI-TOF/R
4. Compare observed masses with masses in databases obtained from virtual tryptic digest of all proteins
5. Confidence for hits depends on coverage : minimum 5 masses
Proteomics with MALDI-TOF/R
Complications• Noise
– Due to contamination and other reasons• Low signal
– Insufficient sample, poor digestion, poor extraction– Contaminants that affect ionization: SDS, acrylamide, salts, detergents, PEG
• Miss-cleavage– RPSDPSYKVHRYAKSGGS– VHRYAK may be present in the result
• Half-tryptic peptides– The peptide may break at a non-tryptic site, for some reasons. E.g.
between D-P• Absence of peptides
– Due to various of reasons• False positives.
– By chance a spectrum matches a protein in a database.
10 ppm
1 ppm
10 ppm
1 ppm
Yeast
C. Elegans
Calculated percentage “uniqueness” for masses 500-4,000
0.1 ppm
0.1 ppm
Most Peptides Do Not Have Unique Mass!
Further reduce mass ambiguity
• Use other information about the peptides– Such as retention time.