Download - Faster Mass Spec - worldwide.promega.com
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Michael M. Rosenblatt, Ph.D.
Faster Mass Spec:
Same-Day Sample Prep Now a Reality
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Drug Discovery
Biomarker Discovery
Protein Structure (HDX Mass Spec)
Subcellular Localization
Protein ExpressionProtein Interactions
Chemical Proteomics
Biologics
Applications of Mass Spec in Biology
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Why Trypsin?…Multiple Reasons!
Average peptide size between 700-1500 Da (ideal for MS)
All peptides have a C-terminal charge (due to K/R)
Highly active
Highly specific
Autolysis can be controlled by lysine/arginine modification
Protein
Proteolysis
Mass spec analysis
Peptides
Peptide Sequence
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The Challenges of the Digestion Workflow
• Proteases are the cornerstone of
the bottom-up proteomics workflow
• Traditional protocols require 4-18
hours of digestion
• Reduction/alkylation are often
required to increase sequence
coverage and ensure complete
digestion
• Chemical denaturants inhibit
proteolysis and may require offline
desalting steps
Prepare Sample
DenatureReduce Alkylate
Trypsin Digestion
Step Timing
Hours to days
2 hours
Typically overnight Would reducing sample prep time and
simplifying the workflow be helpful?
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Heat as an Alternative to Chemical Denaturation
Using heat as the denaturant has two advantages:
1. Increases the enzymatic activity
2. Requires no chemical additives, resulting in cleaner samples.
Heat: • Speeds up proteolysis
Denaturants: • Slows/Inhibits proteolysis
http://www.intechopen.com/books/abiotic-stress-plant-responses-and-applications-in-agriculture/abiotic-stress-adaptation-protein-folding-stability-and-dynamics
The Benefit: Proteins unfold, exposing buried proteolytic sites for digestion.
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Rapid Digestion Workflow
Protein Sample
Add Rapid Digestion
Buffer
Add Protease -
Heat (70°C)
LC- MS/MS Analysis
Results
RT: 0.00 - 70.00 SM: 7B
0 5 10 15 20 25 30 35 40 45 50 55 60 65
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
27.27
15.64
21.6816.42
20.99 34.2619.08 32.8913.46
40.17
23.18
38.9327.93
26.45 49.15
41.4625.95
12.75
46.38
36.80
69.76
69.55
53.45 69.38
69.2554.08
11.64
68.14
51.43 67.6742.599.7857.10
9.110.23 59.99 64.816.00 61.424.15
NL: 3.70E9
m/z= 55.6667-6000.0000 F: FTMS + p NSI Full lock ms [400.00-2000.00] MS 09012015_QC_15
+
HeatProtease
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Rapid Digestion Proof of Concept:Digestion of Insulin in 30 Minutes
C = Undigested Insulin A = Small FragmentB = Large Fragment
Retention Time (min.)
A2
20
A
B
Undigested Insulin
Undigested Insulin
Rapid Trypsin30 minutes, 70°C
Conventional Digest18 hours, 37°C A
B
A B
Cleavage Site
C
C
Rapid Trypsin completes digestion in 30 minutes
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Time Course of Proteolysis with Rapid Trypsin
Digestion is essentially complete in 20 minutes!
Undigested Insulin
Small Fragment
LargeFragment
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0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Seq
ue
nce
Co
vera
ge
Rapid Trypsin Overnight Control
Rapid Digestion of Multiple Protein Substrates
Equivalent or better sequence coverage with Rapid Trypsin compared to standard digestion
Overnight Control• 37°C, 50 mM AmBic.
Rapid Trypsin• 70°C, 30 minutes
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0
200
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600
800
1000
1200
1400
1600
1800
PSM
's
Rapid Trypsin OvernightControl
Rapid Digestion of Multiple Protein Substrates
Overnight Control• 37°C, 50 mM AmBic.
Rapid Trypsin• 70°C, 30 minutes
Equivalent or better PSMs with Rapid Trypsin compared to standard digestion
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Digestion of IgG1 with Rapid Trypsin
Digestion of most peptides is complete within 30 minutes
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Rapid Trypsin Gives Robust Quantitation for IgG1 Signature Peptides
Range: 50 ng/mL – 200 mg/mL
y = 0.7219x + 0.2484R² = 0.9999
0
20
40
60
80
100
120
140
160
0 50 100 150 200 250
Re
spo
nse
Rat
io
[mAb] (mg/mL)
ALPAPIEK
y = 0.795x - 0.6049R² = 0.9977
0
20
40
60
80
100
120
140
160
180
0 50 100 150 200 250
Re
spo
nse
Rat
io
[mAb] (mg/mL)
EVQLVESGGGLVQPGGSLR
y = 0.7123x + 0.3008R² = 0.9998
0
20
40
60
80
100
120
140
160
0 50 100 150 200 250
Re
spo
nse
Rat
io
[mAb] (mg/mL)
FNWYVDGVEVHNAK
y = 0.7699x - 0.1147R² = 0.9999
0
20
40
60
80
100
120
140
160
180
0 50 100 150 200 250
Re
spo
nse
Rat
io
[mAb] (mg/mL)
GLEWVSK
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Quantitative Analysis Using a QqQ Instrument
The Rapid Trypsin protocol can be used for quantitative analytical applications. Both precision and
accuracy are within GLP guidelines with strong linearity from 0.1- 25µg/mL. All samples included a
heavy labeled IgG1 at a concentration of 5µg/mL as an internal standard. All data were processed
with Skyline (U. Washington).
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Rapid Trypsin for the Analysis of
Complex Protein Mixtures
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18-Protein Test Mixture
Protein Molecular Weight (Da)
Cytochrome c 12300
Lysozyme 16238
α-Lactalbumin 16246
RNase A 16460
β-Lactoglobulin 19883
Carbonic Anhydrase 29113
GAPDH 35746
Alcohol Dehydrogenase 36849
Ovalbumin 45756
Glutamic Dehydrogenase 61511
Serum Albumin 69366
Apotransferrin 77666
Lactoperoxidase 80642
Phosphorylase B 97289
Beta-Galactosidase 116512
Thryoglobulin 303221
Hemoglobin 64000
Myoglobin 16900
• Protein Test mixture allows for detailed assessment of protein digestion
• Large assortment of sizes and disulfide bond content
• All proteins equal concentration (1 µM)
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Rapid Trypsin Digestion of 18-Protein Mixture15 Minute Digestion Time
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%Se
qu
ence
Co
vera
ge (
%)
37 °C 70 °C
Rapid Trypsin digestion results in high sequence coverage of proteins in 15 minutes.
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Analysis of Individual Proteins in the MixtureTotal Spectra
0
10
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30
40
50
15 30 60 90 120
Tota
l Sp
ect
ra
Time (minutes)
Lysozyme
0
50
100
150
200
15 30 60 90 120To
tal S
pe
ctra
Time (minutes)
Serum Albumin37 °C 70 °C
0
100
200
300
400
500
600
700
800
15 30 60 90 120
Tota
l Sp
ect
ra
Time (Minutes)
Thyroglobulin37 °C 70 °C
0
20
40
60
80
100
120
140
160
15 30 60 90 120
Tota
l Sp
ect
ra
Time (minutes)
Lactoperoxidase37 °C 70 °C
0
20
40
60
80
100
15 30 60 90 120
Tota
l Sp
ect
ra
Time (Minutes)
Myoglobin37 °C 70 °C
0
100
200
300
400
500
600
700
800
15 30 60 90 120
Tota
l Sp
ect
ra
Time Minutes
Beta-Gal37 °C 70 °C
37 °C 70 °C
At 70°C, digestion is complete within 15 minutes for 6 proteins above. At 37°C, many are not fully digested even in 2 hours.
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Rapid Trypsin Generates Fully Digested Signature Peptides within 15-30 Minutes
0
1E+10
2E+10
3E+10
4E+10
5E+10
15 20 60 90 120 180 240
Pe
ak A
rea
Time (minutes)
R.WVGYGQDSR.L (Beta-Gal)70 C 37 C
0
2E+10
4E+10
6E+10
8E+10
15 20 60 90 120 180 240
Pe
ak A
rea
Time (minutes)
K.HGTVVLTALGGILK.K [Myoglobin] 70 C 37 C
0
2E+10
4E+10
6E+10
8E+10
15 20 60 90 120 180 240
Pe
ak A
rea
Time (Minutes)
R.GGLEPINFQTAADQAR.E [Ovalbumin]70 C 37 C
0
1E+10
2E+10
3E+10
15 20 60 90 120 180 240
Pe
ak A
rea
Time (Minutes)
R.WLPAEYEDGLALPFGWTQR.K [Lactoperox]
70 C 37 C
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Rapid Trypsin Digestion of Yeast Extract
1 Hour Digestion
20302 Spectra708 Proteins250 ng analyzed
Overnight Digestion
18053 Spectra721 Proteins250 ng analyzed
Rapid Trypsin
Standard Overnight
Digestion of the complex mixture using the Rapid Trypsin reagent is comparable to the overnight digestion protocol.
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Rapid Trypsin / Lys-C
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In Addition to Rapid Trypsin, There is Rapid Trypsin/Lys-C More Efficient Digestion at Elevated Temperature
Missed R3.6% 4%
Missed K18.6%
NNNNNRNNNNNK NNNNNN
Trypsin cleavage specificity
Trypsin digest
Missed K:Missed R = 5.5:1 Missed K:Missed R ~ 1:1
Trypsin/Lys-C digest
3.6%
NNNNNRNNNNNK NNNNNN
Trypsin/Lys-C cleavage specificity
Trypsin/Lys-C eliminates majority of missedlysine sites and improves overall digestion efficiency.
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Fewer Missed Cleavages with Rapid Trypsin/Lys-C Mixture
All Samples 1 hour digestion
65
70
75
80
85
90
95
Rapid Trypsin Rapid Trypsin /LysC
Rapid Trypsin Rapid Trypsin /LysC
Rapid Trypsin Rapid Trypsin /LysC
Fully
Cle
ave
d P
ep
tid
es
(%)
Lysine Digestion Efficiency
Arginine Digestion Efficiency
Total Efficiency
18 Protein Mixture Yeast Human
2426 Proteins
2240Proteins
721Proteins
708Proteins
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Rapid Trypsin/Lys-C Improves Sequence Coverage for “Difficult” Proteins
Rapid Trypsin(57 % coverage)
Rapid Trypsin/ Lys-C(76 % Coverage)
Beta-Galactosidase
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Rapid Trypsin/Lys-C for Quantitation:Improved Precision
0
5
10
15
20
25
30
35
40
45
50C
V (
%)
(N=1
0 r
eplic
ates
)
Rapid Trypsin Rapid Trypsin/ LysC
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Rapid Trypsin is Compatible with IgG
Analysis from Serum
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Workflow Compatibility: IgG Enrichment
Binding
Wash
Elute
Neutralize
Enriched target antibody + internal standard
Add internal standard
+
Plasma/serum
Wate
r_R
T
TF
A_R
T
Wate
r_R
TL
C
TF
A_R
TL
C
0
5
1 0
1 5
2 0
2 5
A L P A P IE K
Re
sp
on
se
Ra
tio
Rapid Trypsin Rapid Trypsin/Lys-C
Water TFA Water TFA
Peak areas of signature peptides are also unchanged and very precise
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Rapid Trypsin Can Be Used with or
without Reduction and Alkylation
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Two Approaches to Using Rapid Trypsin
Direct Approach (Most Substrates)
1. Obtain Protein sample
2. Add 3X volume of Rapid Digestion Buffer (RDB)
3. Add Rapid Digestion Enzyme (Rapid Trypsin or Rapid Trypsin/Lys-C)
4. Incubate (15 min to 2 hours) – depending of substrate
5. Quench with neat formic acid (5 microliters per 0.2 mL)
6. Direct LC-MS/MS analysis
Reduction Alkylation (Disulfide-rich or Difficult to Digest Substrates)
1. Obtain Protein Sample
2. Add Rapid Digestion Buffer (RDB)
3. Reduce with up to 2 mM TCEP and alkylate with 2 mM IAM (reduction alkylation can
be done prior to digestion or concurrent)
4. Steps 3-6 above
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Reduction/Alkylation Can Be Used to Improve Sequence Coverage
Non-reduced Reduced
RapidTrypsin
RapidTrypsin/Lys-C
RapidTrypsin
RapidTrypsin/Lys-C
For disulfide-rich proteins, reduction and alkylation improves sequence coverage.
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Reduction/Alkylation can be Performed Simultaneously with Rapid Digestion
0
10
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60
70
80
# o
f U
niq
ue
Cys
tein
e C
on
tain
ing
Pe
pti
de
s
Rapid Trypsin_PriorReductionRapid Trypsin with LysC_PriorReductionRapid Trypsin_Simultaneous ReductionRapid Trypsin with LysC_Simulatneous ReductionRapid Trypsin_no ReductionRapid Trypsin with LysC_no Reduction
A panel of disulfide-rich proteins all show, in most cases, similar or more unique cysteine-containing peptides produced in a simultaneous Red/Alk protocol versus a prior Red/Alk step
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Customer Data
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Evaluation of Complex Mixtures Using Rapid Trypsin/Lys-C: Rapid Digestion is Comparable to Overnight Digestion
Gutierrez, D.B., et al. & Caprioli, R.M. Validation of a unified sample preparation platform for multi-omics technologies. 64th Conference on Mass Spectrometry and Allied Topics. ASMS. San Antonio TX, June 2016.
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Analysis of Membrane Proteome Fractions
0
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900
mem 1 mem 3 mem 2 mem RT 1 mem RT 2 mem RT 3
#Prot
0
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16000
18000
mem 1 mem 3 mem 2 mem RT 1 mem RT 2 mem RT 3
#Peptide IDs
Regular protocol Rapid Trypsin protocol Regular protocol Rapid Trypsin protocol
The Rapid Trypsin protocol yields more proteins and peptides at
comparable LC-MS/MS settingsDr. Andreas Otto – University of Greifswald
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Analysis of Membrane Proteome Fractions
Regular
protocol
Rapid Trypsin
protocol
16876647
25124640971 unique peptides
unique spectra
proteins
326256741362
Dr. Andreas Otto – University of Greifswald
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Complete Coverage of the TM Domain
ARTQ_BACSU Rapid Trypsin ARTQ_BACSU regular in solution digest
Dr. Andreas Otto – University of Greifswald
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C.
B.
A.
Tru
e tr
ypti
cM
iscl
eave
dQ
uad
rup
let
acet
ylat
ed
Histone Sequence Coverage and PTMs
The short digestion times are critical for identifying and confirming histone modified peptides
Rapid Digestion of Histones with TMT Labeling
Chris Adams - Stanford
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Rapid Digestion Protocol
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Rapid Digestion Protocol
Step Timing
Obtain Protein –add digestion buffer
1 minute
Add Enzyme 1 minute
Incubate at temperatures up to 70°C
15 minutes to several hours
Add quench agent (formic acid)
1 minute
Analyze by LC-MS/MS 30 minutes to several hours
Key Points:
• Fast
• No chemical denaturants required
• Reduction/Alkylation not required but can be used if necessary
• No vigorous shaking needed
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Kit Features
Kit components:
• Rapid Digestion Buffer – Optimized for proteolysis at elevated temperatures (70°C)
and short digestion times.
• Rapid Trypsin Enzyme or Rapid Trypsin/Lys-C mixture
• Enzyme Resuspension Buffer
Advantages:
• Reduction and Alkylation not required
• Enzyme is not immobilized and therefore no need for offline filtration or desalting
• Compatible with workflows that involve surfactants and/or denaturants
• Short workflow – with digestion times as short as 15 minutes, LC-MS/MS results can
be produced within several hours (from samples to search results)
Product is currently available for early access sales – contact your local
Promega Rep or call our sales desk for details.
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Conclusions
• Proteolysis of protein substrates can be accomplished in as little as
15 minutes with Rapid Trypsin.
• Rapid Trypsin / Lys-C is beneficial for quantitative applications (like
drug metabolism) and also for complex mixtures (like lysates).
• The Rapid Trypsin protocol is compatible with Reduction/Alkylation,
denaturants, as well as affinity enrichment.
• The flexibility of Rapid Trypsin makes it highly amenable for analysis
of a broad range of samples including membrane proteins, complex
mixtures, as well as TMT labeling.
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Acknowledgements
• Marjeta Urh, Ph.D.
• Gary Kobs
• Sergei Saveliev , Ph.D.
• Chris Hosfield, Ph.D.
• Katiria Rivera
• Brett Schumacher
• Caprioli Lab – Vanderbilt University - MSRC (Danielle Gutierrez Ph.D., Carrie Romer,
Jeremy Norris, Ph.D.)
• Stanford Proteomics Core – Dr. Chris Adams
• University of Griefswald – Prof. Dr. Dorte Becher, Dr. Andreas Otto and Minia Antelo
(Department of Microbial Proteomics and Mass Spectrometry)
• Merck (West Point, PA) – Kevin Bateman, Ph.D and Daniel Spellman, Ph.D.
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For more information, contact
Michael Rosenblatt
For Early Access, contact
Gary Kobs, Global Product
Manager
Thank You