1. Mass Fingerprint

2. 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.enzymeproteolysiscatabolismhydrolysis 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-RQ-V-…

3. R- G- F - K- I - A - E - W - M  MW (Average mass):  1136 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-  R n-1 R n R n+1 Trypsin cleaves at only at peptide bond R n-1 = K, R; Rn  P Note: each internal peptide will end with Lys (K) or Arg (R) Example:  Cleavage with Trypsin (tryptic digestion)

4. 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.

5. internal calibrants Mass “Fingerprint” of a Pure Protein Peptides from trypsin self-digestion MALDI-TOF/R MS of Peptides from a Tryptic Digest

6. Search a database for match RPSESSYKVHRYAKSGGSanother protein…… in-silicon digestion in-silicon digestion ……

7. 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

8. Mass Accuracy is Important

9. 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.

10. Mascot interface

11. 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

13. 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.

14. 10 ppm 1 ppm 10 ppm 1 ppm Yeast C. Elegans Calculated percentage “uniqueness” for masses 500-4,000 0.1 ppm Most Peptides Do Not Have Unique Mass!

15. Further reduce mass ambiguity Use other information about the peptides – Such as retention time.