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

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Page 1: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

Mass Fingerprint

Page 2: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

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-…

Page 3: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

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)

Page 4: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

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.

Page 5: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

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

Page 6: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

Search a database for match

RPSESSYKVHRYAKSGGS another protein ……

in-silicon digestion

in-silicon digestion

……

……

Page 7: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

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

Page 8: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

Mass Accuracy is Important

Page 9: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

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.

Page 10: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

Mascot interface

Page 11: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

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

Page 12: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that
Page 13: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

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.

Page 14: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

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!

Page 15: Mass Fingerprint. Protease A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that

Further reduce mass ambiguity

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