Trypsin

Question 1

Discuss how trypsin is central to the preparation of peptides for mass spectrometry analysis and strategies that are availible when alternatives to trypsin are needed.

Answer 1

1. Why proteolysis is needed, methods of denaturation
2. why trypsin is optimal, how it works
3. proteolysis (challenges, solutions and limitations)
4. when trypsin is not enough, alternative proteases
5. chymotrypsin and thermolysin

Question 2

1. Why proteolysis is needed, methods of dentaturation (physical and chemical)

Answer 2

1. Chaotropes (urea and thiourea)
   - solubilises and unfolds most proteins to their fully random confirmation by disrupting hydrogen bonds (very important for protein folding)
   - all ionisable groups are exposed to solution
   - urea conc = 5-7M, thiouea conc = 2M
2. Detergents
   - ionic, non-ionic or zwitterionic detergents e.g SDS
   - type used dependant on type of protein your trying to solubilise
   - SDS = anionic detegent that disrupts non-covalent interactions in native proteins., ensures complete sample solubilistation and prevents aggregation through hydrophobic interactions (breaking hydrophobic interactions that form and preventing aggregation, breaking the bonds that keep these proteins folded)
3. Reducing Agent
   - e.g DTT
   - breaks disulfide bonds that form between cystine residues (keep protein folded)
   - maintain proteins in their fully reduced state
   - other alternatives include beta-mercaptoethanol
4. Physical methods
   - dounce pestels, sonication, homogenisation
5. Why proteolysis is necessary?
   - denaturation unfolds protein but still too big to be analysed by mass spectrometer
   - range of masses is limited in the typical mass spec (250-4000 daltons)
   - soolution = site specific endoproteinases

Question 3

2. Why trypsin is optimal and how it works

Answer 3

1. facts about trypsin that make it optimal
   - known as the gold standard for bottom up proteomics
   - digestive protease, produced in the pancreas of porkine pigs, component of pancreatic juice and is released in the duodenum during digestion
   - left to digest protein overnight at 37 degrees celsius as that is it’s physiological potential i.e temperature at which pancreatic juice works
   - average size of generated peptides is between 700 - 1500 daltons which is ideal for mass spec analysis
   - all generated peptides have a c-terminal charge as it was cleaved at lysine/arginine
   - par consequence, highly active (works in a large number of different chemicals) /highly specific (only cleaves at lysines or arginines
2. how it works
   - as lysine and arginine are basic amino acids, their side chain have a positive charge
   - trypsin active site contains a negatively charged aspartate residue that interacts with this positive charge and cleaves at the carboxylic side of arginine/lysine residues
   - this results in each generated peptide ending with either a lysine/arginine and having a positive charge
   - this positive charge is essential as it allows the peptide to move around within the instrumet and hit the detector for detection and quantification
   - the sizes of the peptide fragments obtained after trypsin digestion, represent the peptide mass fingerprint and are characteristics of each protein (barcode of peptides)

Question 4

3. Proteolysis with trypsin (Challenges, solutions and limitations)

Answer 4

1. Challenges
   - protein folded too tightly so trypsin can’t access
   - protein insoluble and requires additives
2. Solutions
   - denaturing agents (urea, guanidine HCL and organic solvents)
   - detergents e.g triton X-100 and SDS (increasing the concentration)
3. Limitiations
   - protease inhibition, increasing the concentration of chemicals starts to inhibit trypsin (too many chemicals) stops its activity
   - unwanted side effects e.g protein modification, methionine can become oxidized which adds an additional mass on peptide due to oxidization
   - detergents hurt the hardware, mass spec instrument reduces sensitivity/accuracy, barrier between detector and peptides of interest

Question 5

4. When trypsin is not enough, alternative proteases

Answer 5

sometimes even with other measures undertaken trypsin is not enough to generate peptides

• substantial number of tryptic peptides too long/too short for mass spec analysis depending on the amount of lysine/arginines present in the protein
• tryptic cleavage sites might not be accessible due to post translational modifications such as phosphorylation, glycosylation, histone methylation/acetylation which could cause steric hinderance
• certain proteins not efficiently digested by trypsin e.g membrane proteins and proteins in tight conformations

solution = alternative proteases, chymotrypsin and thermolysinn which allow for more sequence coverage and protein ID’s

Question 6

5. Chymotrypsin and Thermolysin

Answer 6

1. Thermolysin
   - thermostable metalloproteinase
   - preferentially cleaves at the N-terminus of the hydrophobic residues leucine, phenylalanine, valine, isoleucine, alanine and methionine (depends on the nature of the protein/buffer as it is hard to predict)
   - optimal digestion temperature range is 65 - 85 degrees celsius
   - optimal activity at pH 5.0 - 8.5
2. Chymotrypsin
   - serine endoproteinase derived from bovine pancreas
   - preferentially hydrolyzes at the carboxyl side of aromatic amino acids; tyrosine, phenylalanine and tryptophan
   - activity optimal at pH 7.0 - 9.0
   - in an experiment when used in conjuction with trypsin to digest a cellular lysate, it increased the proteome coverage of cells form 3313 identified proteins to 3908 identified proteins