ACCELERATING DIGESTION OF PROTEOMES
USING TRYPSIN-IMMOBILIZED MAGNETIC
NANOPARTICLES
Christie Hunter1, Uma Kota2, Sean Seymour1, Mark Stolowitz2
1AB
SCIEX, USA; 2Canary Center at Stanford for Cancer Early Detection, Dept. of Radiology, Stanford School of Medicine, Palo Alto, CA, USA.
Figure 2: Comparison of Yield from the Different Digestion Strategies. 20 μg of E.coli cell
lysate was denatured, reduced and alkylated followed by digestion with soluble trypsin (E/S of
1:20) and 300 μg of TIMPs with or without microwave irradiation. Samples were randomized and
analyzed in duplicate by LC/MS/MS. This plot shows the false discovery rates (FDR) at critical
threshold values for both global and local error rate calculations at the protein, distinct peptides
and spectral level indicating that in-solution digestion gives better yield compared to immobilized
trypsin. The poor peptide yield from solid phase-based digestion methods could be due to nonspecific sample absorption and/or protein aggregation on the TIMPs.
A. In solution digestion with TPCK-treated
trypsin
Comparison of the two denaturants relative to each other when the same enzyme was used
shows very little difference between using RapiGest or OGS (RapiGest providing ~10% more
peptides).However, comparing the two different types of trypsin shows that Promega trypsin
produces fewer missed cleavages across all time points with little difference seen in the
percentage of over-digestion components.
A
B
TIMPs + Microwave 90 sec-Rep 2
TIMPs + Microwave 90 sec-Rep 1
TIMPs 6 h-Rep 2
TIMPs 6 h-Rep 1
The relative efficiency of trypsin digestion under the many different conditions
tested here was measured in terms of yield at the peptide and protein level,
percentage canonical sequences, missed cleavages and over-digestion
products as well as artifacts formed during the course of in-solution digestion.
Solution 6 h-Rep 2
Mass spectrometry (MS)-based protein characterization is typically
performed using the shotgun approach where proteins are subjected to
enzymatic digestion or chemical hydrolysis to generate peptides.
Reproducible, rapid and complete digestion of proteins in complex mixtures
is crucial for in-depth protein identification and quantification in bottom-up
proteomics. Conventional in-solution tryptic digestion is time-consuming and
prone to artifacts, reducing dynamic range of MS detection in complex
samples. Immobilized trypsin has several advantages, allowing higher
protein to enzyme ratio, small sample volume, easy sample manipulation,
limited autolysis products and shorter incubation times. Immobilized enzyme
on magnetic particles offers a distinct advantage over soluble enzyme as
they serve both as absorbers of microwave radiation that accelerates the
digestion process and affinity probes that facilitate the separation of enzyme
from products by using an external magnetic field.
There are several different types of commercially available trypsin and
numerous digestion procedures cited in literature. The objective of this study
is to evaluate the relative digestion efficiency and fidelity of the different
types of trypsin (modified, unmodified) and digestion strategies (in solution,
solid phase and microwave-assisted). In addition to measuring protein and
peptide yield, this study also analyzes the percentage of canonical
sequences, missed cleavages and over-digestion products under different
conditions over a time course with a goal of finding the optimal conditions for
quantitative MS studies.
The combination of the technologies employed here is particularly well
suited to do this type of assessment. First, the depth of acquisition provided
by the TripleTOF™ 5600 System makes it much more likely to observe
multiple state variants of the desired canonical peptide, including partitioning
to low stoichiometry species. Secondly, the combination of the use of
sequence tags in the Sequence Temperature Value approach and use of
feature probabilities to structure search space allows the Paragon™
Algorithm in ProteinPilot™ Software to look for very large numbers of
digestion and modification variations without the usual loss in identification
discrimination. The ProteinPilot Descriptive Statistics Template enables easy
distillation of the massive amounts of identification data into informative plots
and tables.
Solution 6 h-Rep 1
RESULTS
Percent deviation versus Reference
INTRODUCTION
B. In solution digestion with
unmodified trypsin
C
Figure 6: Characterization of Sequencing Grade Promega Trypsin. Analysis of the
peptide yield (A) and canonical analysis done in Figure 6, indicate that the 3 hr digestion time
point with either OGS or RapiGest produced a digest of highest fidelity. The breakdown of
these peptide components as a function of precursor signal intensity is shown (B) and the
fraction of peptides that are canonical (C) is plotted. As expected, there is a decrease in the
fraction of peptides that are canonical as the less abundant proteins (measured as precursor
signal) in the sample are analyzed.
METHODS
Preparation of Trypsin Immobilized Magentic Particles (TIMPs): Unmodified
porcine pancreatic trypsin (USBioAnalyzed/Affymetrix) was immobilized onto
amine-functionalized superparamagnetic particles (BcMag amine- terminated
magnetic beads,1 µm ; Bioclone Inc.) by cross-linking the primary amines of trypsin
and the particles using bis-succinimidyl ester-activated PEG compounds. The amount
of trypsin immobilized onto the magnetic particles was determined by measuring the
protein amount in solution before and after the immobilization reaction using BCA
assay.
Comparsion of Different dDigestion Strategies: E.coli cell lysate was denatured
using 1% N-octylglucoside (OGS), reduced with 50 mM TCEP and alkylated with
100mM iodoacetamide. In solution digestion was done using Proteomics grade trypsin
(TPCK-treated, Sigma) at an enzyme: substrate ratio (E/S) of 1:20 at 37 ºC across 4
time points. Solid-phase digestion was done using 300 μg of TIMPS at
37 ºC and monitored across 5 time points. Microwave- assisted solid phase digestion
was carried out using a domestic microwave with output power of 1200 W.
Comparison of Different Types of Denaturants and Trypsin: 293T cell lysate was
denatured using either 1% N-octylglucoside (OGS) or 0.1 %RapiGest followed by
reduction and alkylation as above. In-solution digestion were done using either
Promega Sequencing Grade Modified Trypsin (Trypsin 1) or Sigma Proteomics grade
Trypsin (Trypsin 2) at an E/S of 1: 20 at 37 ºC. The digestion was monitored every 10
min up to 60 min followed by 90 min, 3h, 6h and 16h. The samples were prepared in
triplicate on three separate days.
Figure 3: Comparison of Digestion Efficiency between TPCK-treated and Unmodified
Trypsin. In the case of TPCK-treated trypsin digestion (A), the percentage of canonical
sequences shows a decrease at all time points, earlier time points were then used in subsequent
studies. As digestion proceeds overnight, the percentage of canonical and under-digested
peptides decreases with a concurrent increase in the percentage of over-digested products. A
similar trend is seen for unmodified trypsin (B) in all three categories, however the percent over digested peptides is much higher than the TPCK-treated trypsin at 1 h and continues to
increases with longer digestion time. This drastic increase in over-digested products can be
attributed to the chymotrypsin activity in the untreated trypsin as measured in Figure 4.
A. Digestion frequencies for
TPCK-treated trypsin
B. Digestion frequencies for
unmodified trypsin
Chromatography: The sample was analyzed using the Eksigent nanoLC-Ultra® 2D
System plus combined with the cHiPLC®-Nanoflex system configured in trap elute
mode (Eksigent, Dublin, USA). The cell lysate (1 ug total protein) was loaded at
2 μL/min onto a 200 µm x 0.5 mm precolumn and eluted at 300 mL/min onto a
analytical 75 µm x 150 mm column. Both pre-column and analytical column were filled
with ChromXP C18-CL 3μm 120 Å phase (Eksigent). An elution gradient of 10-35%
acetonitrile (0.1% formic acid) with a 60 min gradient was used.
E. coli cell lysate
Human Cell Lysate
1% N-octylglucoside (OGS)
1% N-octylglucoside
Reduction/ Alkylation
Reduction/ Alkylation
Reduction/ Alkylation
Dilute
Dilute
Dilute
TIMPs
digestion at
37°C
5 time points
TIMPs +
microwaveassisted
6 time points
In-solution
digestion
4 time points
Sigma
trypsin
In-solution
digestion
10 time points
Sigma
Trypsin
In-solution
digestion
10 time points
Promega
Trypsin
0.1% Rapigest
In-solution
digestion
10 time points
Sigma Trypsin
In-solution
digestion
10 time points
Promega
Trypsin
Figure 4: Digestion Map Comparing the Cleavage Frequency by Residue Pair for the
Different Grades of Trypsin. Matrix view of the digestion frequency between each pair of
residues for E.coli cell lysate digested in solution with TPCK-treated trypsin (A) or unmodified
trypsin (B). Untreated trypsin has chymotrypsin activity that cleaves after Leu (L), Phe (F), Trp
(W) and Tyr (Y) residues. The chymotrypsin activity in the untreated trypsin as seen in 4(B)
explains the higher percentage of over-digestion observed in Figure 3B.
OGS / Promega Trypsin
OGS / Sigma Trypsin
• Analysis of digestion time courses concurrent with the analysis of
canonical peptides and their under- and over- digested counterparts
is key to understanding and optimizing digestion for quantitative MS
assays.
• Use of lower grade trypsin that has not been TPCK treated shows a
dramatic increase in over-digestion as expected. Similarly, longer
digestion times can produce an increase in the sample preparation
artifacts and therefore may be less desirable than previously
believed.
• Proteomics grade trypsin from Sigma showed a higher rate of
missed cleavages across the time points suggesting that perhaps
this enzyme had lower activity or required a higher protein to enzyme
ratio (needs to be investigated).
• N-Octylglucoside and RapiGest provided similar profiles in terms of
proportion of digestion artifacts observed, with RapiGest providing a
slightly higher yield of peptides (~10%). The current data do not
enable a definite conclusion about the relative performance of the
two denaturants.
RapiGest / Promega Trypsin
RapiGest / Sigma Trypsin
• Detailed characterization of sample preparation strategies are critical
for MS quantitative studies, it must be reproducible and have high
fidelity especially in targeted proteomics studies where absolute
quantitative values are desirable.
ACKNOWLEDGEMENTS
LC/MS/MS Analysis
This research is funded by the Canary Foundation. www.canaryfoundation.org
We would like to thank Ken Lau for help with the sample preparation.
ProteinPilot™ Software Database Search
Figure 1. Overview of Experimental Design. Analysis of all samples was performed in the
same way. Steps shown in grey boxes were held constant. Colored boxes indicate the
parameters that were varied.
• Multiple digestion strategies and reagents were evaluated for their
yield and fidelity.
• Use of Trypsin Immobilized Magnetic Particles (TIMPs) and the
combination with microwave digestion shows a loss in peptide yield,
possibly due to surface adsorption of some proteins/peptides to the
magnetic particles or aggregation of proteins at high temperatures
caused by microwave irradiation. Further improvements to the
design and synthesis of TIMPs will be performed.
Quench Reaction
ProteinPilot Descriptive Statistics Template Analysis
CONCLUSIONS
• The combination of deep acquisition with the TripleTOF™ 5600
system and extensive identification provided by the analysis with the
Paragon™ Algorithm enable this level of interrogation in a way that
has not been possible before.
Mass Spectrometry: Data was acquired on the TripleTOF™ 5600 System in IDA
mode using an acquisition rate of 20 MS/MS per cycle x 50 msec minimum
accumulation time. High sensitivity MS/MS mode was used so MS/MS spectra had
resolution >15,000.
Data Processing: All data was processed using ProteinPilot™ Software 4.0, using the
integrated false discovery rate analysis. Results were then analyzed using the PSPEP
Comparison Template and the ProteinPilot Descriptive Statistics Template with some
additional canonical sequence analysis capabilities added in.
Figure 7: Analyzing Sample Preparation Artifacts Produced During Digestion Time
Courses. In addition to the canonical peptide analysis, we looked at the increase in sample
preparation modifications that could occur during digestion. Data shows a steady conversion
of N-terminal Glu residues to pyro-Glu. Minimal changes are seen in the other sample
preparation modifications analyzed. These trends looked similar between the four digestion
conditions discussed in Figure 5, data shown here is from the Promega Trypsin / RapiGest
digestion.
Figure 5: Comparison of Different Digestion Reagents for Impact on Canonical
Peptides. Modified trypsin from different vendors in combination with commonly used
denaturants (OGS and RapiGest) was used to digest human cell lysate to understand their
effect on the quality and efficiency of digestion. (Continued above)
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