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www.promega.com
Missed R Missed R
Missed K (80-90% of missed
cleavages)
Missed K
Marjeta Urh1, Sergei Saveliev1, Ethan Strauss1, Mike Rosenblatt1, Richard Jones2, Michael Ford2
and Dave Allen2
1Promega Corporation, Madison, WI; 2MS BioWorks, Ann Arbor, MI
1. Introduction 2. Text goes here
It can be 2 lines
3. Text goes here
It can be 2 lines
6. Text goes here
It can be 2 lines 2. Proteolytic enhancement with Trypsin/Lys-C
3. Trypsin/Lys-C advantage for mass spec analysis 4. An additional advantage: digestion of
proteolytically resistant proteins
5. Conclusions
Incomplete proteolysis in trypsin digests is largely attributed
to trypsin proteolytic inefficiency at lysine sites. Missed K and missed R ratio in tryptic digests equals to 5:1-6:1
Supplementing trypsin with Lys-C leads to elimination of the
majority of missed lysine sites and overall increase in
proteolytic efficiency.
Trypsin/Lys-C mix tolerates protease inhibiting impurities
and digests tightly folded, proteolytically resistant proteins* *Digestion of proteolytically resistant proteins requires a specialized, two-
step procedure
Enhanced digestion with Trypsin/Lys-C provides critical
benefits for protein mass spec analysis: Increase in peptide and protein identifications
Higher analytical reproducibility
More accurate protein quantitation through more complete digestion
Enhanced protein mass spectrometry analysis with Trypsin/Lys-C mix
Efficient proteolysis is required for protein mass spectrometry
analysis. Here we describe a Trypsin/Lys-C mix, which dramatically
improves proteolysis:
the majority of missed cleavages, commonly occurring in trypsin
digests, are largely eliminated
protease-inhibiting impurities are tolerated
proteolytically resistant proteins are efficiently digested
We show here how enhanced proteolysis with Trypsin/Lys-C
improves protein mass spectrometry analysis. These benefits include
an increase in identified peptides and proteins, higher signal intensity
of individual peptides and higher analytical reproducibility.
Digested sample Missed K
Missed R
Missed K: Missed R
ratio
Yeast extract 18.6% 3.6% 5.5:1
Mouse extract 6.6% 1.1% 6:1
In a typical trypsin digest,
10-30% cleavage sites are
‘missed’ (undigested).
Majority of missed
cleavages are lysines.
Add trypsin/Lys-C Add Urea to 6-8M Dilute the reaction and continue
digestion
Digestion of proteolytically
resistant protein (myoglobin)
with Trypsin/Lys-C
Digestion with Trypsin
(myoglobin remains intact)
Digestion with Trypsin/Lys-C
(myoglobin is efficiently digested)
Intact
protein
Peptides
RP HPLC spectra
Protein
denatures. It is
now amenable to
proteolysis
Lys-C digests a protein
onto relatively large
fragments. Trypsin is
inactivated.
Trypsin re-activates
and completes
digestion
Protein is
resistant to
trypsin due to
tight
conformation
Alternative protocol; a two step digestion.
24% and 10% increase of identified peptides and proteins, respectively.
40% and 17% increase of identified
peptides and proteins, respectively.
NNNNNK NNNNNR NNNNNN
Trypsin/Lys-C cleavage specificity
705 887
Trypsin digest
Trypsin/Lys-C digest
FFPE skin tissue
Identified peptides
1490 2092
Arabidopsis extract
Protein
Protein mass spec analysis
Peptides
Trypsin/Lys-C eliminates the
majority of missed lysine
cleavages and increases
overall proteolytic efficiency.
Digested sample
Missed K
Missed R
Missed K: Missed R
ratio
Yeast extract 2.6% 4% 0.65:1
Mouse extract 2.9% 1.5% 1.9:1
NNNNNK NNNNNR NNNNNN
Trypsin cleavage specificity
0
10
20
IgG (Rituximab) digestion reproducibility
CV peptide peak area (n=10)
SLSLSPGK ALPAPIEK QVQLQQPGAELVK
Trypsin digests (n=10)
Trypsin/Lys-C digests (n=10)
Low CV in Trypsin/Lys-C digest
Increase in peptide and protein identifications
Higher peptide recovery
Trypsin digest
Trypsin digest
Trypsin/Lys-C digest Trypsin/Lys-C
digest 1.7-4.6 increase in peptide peak area. Improved protein quantitation!
Quantitation of Serum Amyloid P component (SAMP) protein in plasma
Higher digestion reproducibility
Improved digestion reproducibility
Tolerance to protease inhibiting agents
0
30
60
1252 1204
1495
1364
Digestion in the presence of protease inhibitors and denaturants Yeast protein extract was used as a model substrate
30%
0%
60%
Missed cleavages Identified proteins
Trypsin digest
Trypsin/Lys-C digest
Efficient digestion in the presence of inhibiting agents accompanied by an increase in protein identifications
10%
0%
20%
Trypsin/Lys-C lowers the level of missed cleavages to norm
The number of identified proteins increases by up to 20%
GuCl, 0.5M Protease inhibitor cocktail, 1x
Courtesy by Dr. Szapacs, GSK
Courtesy by Dr. Adams, Stanford University Courtesy by Dr. Minkoff, University of Wisconsin-Madison
Similar ratios of missed lysine and arginine cleavages sites were observed in Trypsin and Trypsin/Lys-C digests of extracts prepared from other sources including human and E.coli.
Regular one-step overnight digestion with Trypsin
Regular one-step overnight digestion with Trypsin/Lys-C mix
GuCl, 0.5M Protease inhibitor cocktail, 1x
Trypsin digest
Trypsin/Lys-C digest
Identified peptides
Peptide peak area Peptide peak area
Trypsin/Lys-C
CV
peptide p
eak a
rea
(n=
10)
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