Mass Fingerprint

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Mass Fingerprint
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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-
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Cleavage with Trypsin (tryptic digestion)
Trypsin cleaves at only at peptide bond Rn-1 K,
R Rn ¹ P
?
X-X-X-X-X-X-X-X-
Rn-1 Rn R n1
Example
Trypsin
Trypsin
?
?
R- G- F - K- I - A - E - W - M
MW (Average mass) 1136
Treatment with trypsin gives 3 different
fragments  1. R (MW 174)   2. Gly - Phe Lys
(MW 57147128 18 350)  
3. Ile-Ala-Glu-Trp-Met (MW 11371129 186
18 648
Note each internal peptide will end with Lys (K)
or Arg (R)
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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.

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MALDI-TOF/R MS of Peptides from a Tryptic Digest
Peptides from trypsin self-digestion
internal calibrants
Mass Fingerprint of a Pure Protein
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Search a database for match
RPSESSYKVHRYAKSGGS
another protein

in-silicon digestion
in-silicon digestion


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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

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Mass Accuracy is Important
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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.

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Mascot interface
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Proteomics with MALDI-TOF/R

• Cut spots from 2D Gel, destained and tryptic
  digest each spot
• ( Medium to high silver stained spot)
• Extract peptides and purify by ZipTip
• Mix with matrix and analyze by MALDI-TOF/R
• Compare observed masses with masses in databases
  obtained from virtual tryptic digest of all
  proteins
• Confidence for hits depends on coverage
  minimum 5 masses

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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.

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Most Peptides Do Not Have Unique Mass!
0.1 ppm
1 ppm
10 ppm
Yeast
0.1 ppm
C. Elegans
1 ppm
10 ppm
Calculated percentage uniqueness for masses
500-4,000
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Further reduce mass ambiguity

• Use other information about the peptides
• Such as retention time.