Faster Mass Spec: Same-Day Sample Prep Now a Reality. Michael M. Rosenblatt, Ph.D.

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1 Faster Mass Spec: Same-Day Sample Prep Now a Reality Michael M. Rosenblatt, Ph.D.

2 Applications of Mass Spec in Biology Biomarker Discovery Protein Interactions Protein Expression Drug Discovery Subcellular Localization Protein Structure (HDX Mass Spec) Chemical Proteomics Biologics 2

peptide size between 700-1500 Da (ideal for MS) All peptides have a

3 Why Trypsin? Multiple Reasons! Protein Proteolysis Peptides Mass spec analysis Peptide Sequence Average peptide size between 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 3

4 The Challenges of the Digestion Workflow Step Prepare Sample Denature Reduce Alkylate Trypsin Digestion Timing Hours to days 2 hours Typically overnight 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 Would reducing sample prep time and simplifying the workflow be helpful? 4

5 Heat as an Alternative to Chemical Denaturation The Benefit: Proteins unfold, exposing buried proteolytic sites for digestion. Heat: Speeds up proteolysis Denaturants: Slows/Inhibits proteolysis Using heat as the denaturant has two advantages: 1. Increases the enzymatic activity 2. Requires no chemical additives, resulting in cleaner samples. 5

00 SM: 7B 100 95 90 85 80 75 70 13.46 15.

Abundance 26.45 60 49.15 55 50 41.46 25.

45 20 54.08 15 11.64 10 51.43 5 9.78 42.

6 Rapid Digestion Workflow Protein Sample RT: SM: 7B Add Rapid Digestion Buffer Relative Abundance Time (min) + Add Protease - Heat (70 C) Protease Heat LC- MS/MS Analysis Results 6

Rapid Digestion Proof of Concept: Digestion of Insulin in 30 Minutes Undigested Insulin Undigested Insulin C B A 220 Conventional Digest Rapid Trypsin 30 minutes, 70 C A

7 Rapid Digestion Proof of Concept: Digestion of Insulin in 30 Minutes Undigested Insulin Undigested Insulin C B A 220 Conventional Digest Rapid Trypsin 30 minutes, 70 C A 18 hours, 37 C A A B B C C = Undigested Insulin A = Small Fragment B = Large Fragment Cleavage Site Retention Time (min.) Rapid Trypsin completes digestion in 30 minutes 7

8 Time Course of Proteolysis with Rapid Trypsin Undigested Insulin Small Fragment Large Fragment Digestion is essentially complete in 20 minutes! 8

9 Sequence Coverage Rapid Digestion of Multiple Protein Substrates 100% 90% 80% 70% 60% Rapid Trypsin Overnight Control Overnight Control 37 C, 50 mm AmBic. 50% 40% 30% Rapid Trypsin 70 C, 30 minutes 20% 10% 0% Equivalent or better sequence coverage with Rapid Trypsin compared to standard digestion 9

PSM's Rapid Digestion of Multiple Protein Substrates 1800 1600 1400

10 PSM's Rapid Digestion of Multiple Protein Substrates Rapid Trypsin OvernightControl Overnight Control 37 C, 50 mm AmBic. Rapid Trypsin 70 C, 30 minutes Equivalent or better PSMs with Rapid Trypsin compared to standard digestion 10

11 Digestion of IgG1 with Rapid Trypsin Digestion of most peptides is complete within 30 minutes 11

12 Response Ratio Response Ratio Response Ratio Response Ratio Rapid Trypsin Gives Robust Quantitation for IgG1 Signature Peptides ALPAPIEK y = x R² = [mab] (mg/ml) EVQLVESGGGLVQPGGSLR y = 0.795x R² = [mab] (mg/ml) FNWYVDGVEVHNAK y = x R² = [mab] (mg/ml) GLEWVSK y = x R² = [mab] (mg/ml) Range: 50 ng/ml 200 mg/ml 12

13 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 µg/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). 13

14 Rapid Trypsin for the Analysis of Complex Protein Mixtures

15 18-Protein Test Mixture Protein Molecular Weight (Da) Cytochrome c Lysozyme α-lactalbumin RNase A β-lactoglobulin Carbonic Anhydrase GAPDH Alcohol Dehydrogenase Ovalbumin Glutamic Dehydrogenase Serum Albumin Apotransferrin Lactoperoxidase Phosphorylase B Beta-Galactosidase Thryoglobulin Hemoglobin Myoglobin Protein Test mixture allows for detailed assessment of protein digestion Large assortment of sizes and disulfide bond content All proteins equal concentration (1 µm) 15

16 Sequence Coverage (%) Rapid Trypsin Digestion of 18-Protein Mixture 15 Minute Digestion Time 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 37 C 70 C Rapid Trypsin digestion results in high sequence coverage of proteins in 15 minutes. 16

Total Spectra Total Spectra Total Spectra Total

Individual Proteins in the Mixture Total Spectra 50

Lysozyme 37 C 70 C 15 30 60 90 120 Time (minutes)

150 100 50 0 160 140 120 100 80 60 40 20 0 Serum

Lactoperoxidase 37 C 70 C 15 30 60 90 120 Time

60 40 20 0 Thyroglobulin 37 C 70 C 15 30 60 90 120

Time (Minutes) At 70 C, digestion is complete within

17 Total Spectra Total Spectra Total Spectra Total Spectra Total Spectra Total Spectra Analysis of Individual Proteins in the Mixture Total Spectra Lysozyme 37 C 70 C Time (minutes) Beta-Gal 37 C 70 C Time Minutes Serum Albumin 37 C 70 C Time (minutes) Lactoperoxidase 37 C 70 C Time (minutes) Thyroglobulin 37 C 70 C Time (Minutes) Myoglobin 37 C 70 C Time (Minutes) 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. 17

Peak Area Peak Area Peak Area Peak Area Rapid

Peptides within 15-30 Minutes 5E+10 4E+10

L (Beta-Gal) 70 C 37 C 15 20 60 90 120 180

K [Myoglobin] 70 C 37 C 8E+10 6E+10 4E+10

2E+10 3E+10 2E+10 1E+10 R.WLPAEYEDGLALPFGWTQR.

18 Peak Area Peak Area Peak Area Peak Area Rapid Trypsin Generates Fully Digested Signature Peptides within Minutes 5E+10 4E+10 3E+10 2E+10 1E+10 0 R.WVGYGQDSR.L (Beta-Gal) 70 C 37 C Time (minutes) K.HGTVVLTALGGILK.K [Myoglobin] 70 C 37 C 8E+10 6E+10 4E+10 2E Time (minutes) R.GGLEPINFQTAADQAR.E [Ovalbumin] 70 C 37 C 8E+10 6E+10 4E+10 2E+10 3E+10 2E+10 1E+10 R.WLPAEYEDGLALPFGWTQR.K [Lactoperox] 70 C 37 C Time (Minutes) Time (Minutes) 18

19 Rapid Trypsin Digestion of Yeast Extract Rapid Trypsin 1 Hour Digestion Spectra 708 Proteins 250 ng analyzed Standard Overnight Overnight Digestion Spectra 721 Proteins 250 ng analyzed Digestion of the complex mixture using the Rapid Trypsin reagent is comparable to the overnight digestion protocol. 19

20 Rapid Trypsin / Lys-C

In Addition to Rapid Trypsin, There is Rapid Trypsin/Lys-C More Efficient Digestion at Elevated Temperature Trypsin digest Missed K 18.6% 3.6% Missed R 3.

21 In Addition to Rapid Trypsin, There is Rapid Trypsin/Lys-C More Efficient Digestion at Elevated Temperature Trypsin digest Missed K 18.6% 3.6% Missed R 3.6% 4% Trypsin/Lys-C digest Trypsin/Lys-C eliminates majority of missed lysine sites and improves overall digestion efficiency. Missed K:Missed R = 5.5:1 Missed K:Missed R ~ 1:1 Trypsin cleavage specificity NNNNNR NNNNNK NNNNNN Trypsin/Lys-C cleavage specificity NNNNNR NNNNNK NNNNNN 21

2426 Proteins 2240 Proteins 85 80 75 70 65 Rapid Trypsin Rapid Trypsin / LysC Rapid Trypsin Rapid

22 Fully Cleaved Peptides (%) Fewer Missed Cleavages with Rapid Trypsin/Lys-C Mixture 95 Lysine Digestion Efficiency Arginine Digestion Efficiency Total Efficiency Proteins 721 Proteins 2426 Proteins 2240 Proteins Rapid Trypsin Rapid Trypsin / LysC Rapid Trypsin Rapid Trypsin / LysC Rapid Trypsin Rapid Trypsin / LysC 18 Protein Mixture Yeast Human All Samples 1 hour digestion 22

23 Rapid Trypsin/Lys-C Improves Sequence Coverage for Difficult Proteins Beta-Galactosidase Rapid Trypsin (57 % coverage) Rapid Trypsin/ Lys-C (76 % Coverage) 23

24 CV (%) (N=10 replicates) Rapid Trypsin/Lys-C for Quantitation: Improved Precision Rapid Trypsin Rapid Trypsin/ LysC 24

25 Rapid Trypsin is Compatible with IgG Analysis from Serum

internal standard Peak areas of signature peptides are also unchanged and very precise Water TFA

26 R e s p o n s e R a tio Workflow Compatibility: IgG Enrichment Add internal standard + Plasma/ serum Binding A L P A P IE K 2 5 Wash Elute Neutralize Enriched target antibody + internal standard Peak areas of signature peptides are also unchanged and very precise Water TFA Water TFA W a te r _ R T T F A _ R T W a te r _ R T L C T F A _ R T L C Rapid Trypsin Rapid Trypsin/ Lys-C 26

27 Rapid Trypsin Can Be Used with or without Reduction and Alkylation

28 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 28

29 Reduction/Alkylation Can Be Used to Improve Sequence Coverage Rapid Trypsin Rapid Trypsin/ Lys-C Rapid Trypsin Rapid Trypsin/ Lys-C Non-reduced Reduced For disulfide-rich proteins, reduction and alkylation improves sequence coverage. 29

# of Unique Cysteine Containing Peptides Reduction/Alkylation can be Performed Simultaneously with Rapid Digestion 80 70 60 50 40 30 20

LysC_Simulatneous Reduction Rapid Trypsin_no Reduction Rapid Trypsin with LysC_no Reduction A panel of disulfide-rich proteins all

30 # of Unique Cysteine Containing Peptides Reduction/Alkylation can be Performed Simultaneously with Rapid Digestion Rapid Trypsin_PriorReduction Rapid Trypsin with LysC_PriorReduction Rapid Trypsin_Simultaneous Reduction Rapid Trypsin with LysC_Simulatneous Reduction Rapid Trypsin_no Reduction Rapid Trypsin with LysC_no Reduction A panel of disulfide-rich proteins all show, in most cases, similar or more unique cysteinecontaining peptides produced in a simultaneous Red/Alk protocol versus a prior Red/Alk step 30

31 Customer Data

32 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

33 Analysis of Membrane Proteome Fractions #Prot #Peptide IDs mem 1 mem 3 mem 2 mem RT 1 mem RT 2 mem RT 3 0 mem 1 mem 3 mem 2 mem RT 1 mem RT 2 mem RT 3 Regular protocol Rapid Trypsin protocol Regular protocol Rapid Trypsin protocol The Rapid Trypsin protocol yields more proteins and peptides at comparable LC-MS/MS settings Dr. Andreas Otto University of Greifswald 33

34 Analysis of Membrane Proteome Fractions unique peptides proteins unique spectra Regular protocol Rapid Trypsin protocol Dr. Andreas Otto University of Greifswald 34

35 Complete Coverage of the TM Domain ARTQ_BACSU Rapid Trypsin ARTQ_BACSU regular in solution digest Dr. Andreas Otto University of Greifswald 35

36 Quadruplet acetylated Miscleaved True tryptic Rapid Digestion of Histones with TMT Labeling Histone Sequence Coverage and PTMs A. B. The short digestion times are critical for identifying and confirming histone modified peptides C. Chris Adams - Stanford 36

37 Rapid Digestion Protocol

38 Rapid Digestion Protocol Step Obtain Protein add digestion buffer Add Enzyme Incubate at temperatures up to 70 C Add quench agent (formic acid) Analyze by LC-MS/MS Timing 1 minute 1 minute 15 minutes to several hours 1 minute 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 38

39 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. 39

40 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. 40

41 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. 41

com For Early Access, contact Gary Kobs,

42 Thank You For more information, contact Michael Rosenblatt For Early Access, contact Gary Kobs, Global Product Manager 42

Multiplex Protein Quantitation using itraq Reagents in a Gel-Based Workflow

Multiplex Protein Quantitation using itraq Reagents in a Gel-Based Workflow Purpose Described herein is a workflow that combines the isobaric tagging reagents, itraq Reagents, with the separation power