Chapter 8 Sample preparation for mass spectrometry

Question 1

Objectives

Answer 1

Describe the principles of in-gel digestion

Sample preparation for MS

Question 2

Reason for digestion of proteins

Answer 2

Error of mass estimation is proportional to mass of the protein.

Sensitivity of mass measurement increases with the use of smaller peptides (6-20 amino acids)

PTMs further complicate assignments based on mass.

Question 3

How do PTMs complicate assignments based on mass?

Answer 3

Proteases are able to cut proteins at specific amino acid residues to form peptides with a characteristic mass

Question 4

Trypsin introduction

Answer 4

Serine protease of choice

Trypsin is an endopeptidase that usually hydrolyses the peptide bond at the carboxyl side of lysine and arginine

Only exception is when residue P1 is a proline then trypsin is unable to cut the peptide

Question 5

Chymotrypsin

Answer 5

Chymotrypsin is the enzyme that selects for the aromatic amino acids: phenylalanine, tryptophan, and tyrosine

Question 6

Trypsin

Answer 6

Trypsin is the enzyme that selects for the basic amino acids: lysine and arginine.

Question 7

Note about trypsin activity

Answer 7

The activity of trypsin is decreased when acidic residues are present on either side of a susceptible bond. If proline is at the carboxylic side of lysine or arginine, the bond is almost completely resistant to cleavage.

Question 8

Sequencing grade trypsin

Answer 8

To provide maximum specificity

Lysine residues in the porcine trypsin have been modified by reductive methylation, yielding a highly active and stable molecule that is extremely resistant to autolytic digestion.

The specificity of the purified trypsin is further improved by TPCK treatment, which inactivates chymotrypsin. The treated trypsin is then purified by affinity chromatography. It is resistant to mild denaturing conditions such as 0.1% SDS, 1M urea or 10% acetonitrile and retains 50% of its activity in 2M guanidine HCl.

Question 9

Cleavage sites of various proteases

Answer 9

Trypsin - carboxyl side of arginine and lysine residues
LysC - carboxyl side of lysine residues
GluC - carboxyl side of glutamine
AspN - amino side of aspartate residues

Question 10

What does chymotrypsin do

Answer 10

Chymotrypsin hydrolyses the peptide bond mainly at the carboxyl side of tryptophan, tyrosine and phenylalanine and to a lesser extent, the carboxyl side of leucine, methionine and histidine

Question 11

Why is Sequencing grade modified trypsin preferably used in in-gel digestion

Answer 11

Non-modified trypsin can undergo autolytic digestion to form enzyme similar in specificity to chymotrypsin.

Methylated lysine residues on modified trypsin, prevents this self-digestion from occurring.

TPCK (tosyl phenyl chloroketone) is usually added to the digestion to inactivate any residual chymotrypsin-like activity.

TPCK does not affect trypsin.

Question 12

Peptide masses from tryptic digest

Answer 12

Individual peptide mass and the corresponding sequence have been determined through comparison in large databases such as Swissprot and Genbank.

Question 13

8 steps for in-gel digestion

Answer 13

1. Excision of spot(s) from gel(s)
2. Destaining with NH4HCO3 /acetonitrile. It destains
   by reducing hydrophobic and electrostatic
   interaction between the stain and the protein.
3. Reduction with DTT
4. Alkylation with IAA
5. Saturate gel piece with trypsin at 4ºC / on ice (to
   allow enzyme to diffuse into gel).
6. Overnight incubation of trypsin at 37ºC.
7. Extraction of peptides with the following chemicals
   for the respective ionisation methods as part of
   preparation for MS analysis:
   5% formic acid (for protonation of analyte) in
   acetonitrile (a volatile solvent used to solubilise the
   analyte) for electrospray ionisation (ESI)

Trifluoroacetic acid (for protonation and solubilization of analyte) for matrix-assisted laser desorption/ionization (MALDI).

Question 14

Steps in protein digestion

Answer 14

Lysate preparation (lysis, fractionation, depletion, enrichment, dialysis )

Pure protein (in-solution digestion via reduction, alkylation, digestion)

Peptide enrichment/cleanup for MS analysis

Impure proteins are separated by IDE from the pure proteins step and run on gel to separate them

Gel plug is placed in a test tube, continue with in-gel digestion (reduction, alkylation, digestion), peptide extraction back into the peptide step