mass fingerprint. protease a protease is any enzyme that conducts proteolysis, that is, begins...

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.