faster mass spec - worldwide.promega.com

Promega CorporationPromega Corporation©2013 Promega Corporation. Proprietary Information. Not for further distribution.

Michael M. Rosenblatt, Ph.D.

Faster Mass Spec:

Same-Day Sample Prep Now a Reality

2Promega CorporationPromega Corporation©2013 Promega Corporation. Proprietary Information. Not for further distribution.

Drug Discovery

Biomarker Discovery

Protein Structure (HDX Mass Spec)

Subcellular Localization

Protein ExpressionProtein Interactions

Chemical Proteomics

Biologics

Applications of Mass Spec in Biology


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


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?


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.


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


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


Time Course of Proteolysis with Rapid Trypsin

Digestion is essentially complete in 20 minutes!

Undigested Insulin

Small Fragment

LargeFragment


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


0

200

400

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


Digestion of IgG1 with Rapid Trypsin

Digestion of most peptides is complete within 30 minutes


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


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).


Rapid Trypsin for the Analysis of

Complex Protein Mixtures


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)


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.


Analysis of Individual Proteins in the MixtureTotal Spectra

0

10

20

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.


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


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.


Rapid Trypsin / Lys-C


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.


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



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


Rapid Trypsin/Lys-C Improves Sequence Coverage for “Difficult” Proteins

Rapid Trypsin(57 % coverage)

Rapid Trypsin/ Lys-C(76 % Coverage)

Beta-Galactosidase


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


Rapid Trypsin is Compatible with IgG

Analysis from Serum


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


Rapid Trypsin Can Be Used with or

without Reduction and Alkylation


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


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.


Reduction/Alkylation can be Performed Simultaneously with Rapid Digestion

0

10

20

30

40

50

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


Customer Data


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.


Analysis of Membrane Proteome Fractions

0

100

200

300

400

500

600

700

800

900

mem 1 mem 3 mem 2 mem RT 1 mem RT 2 mem RT 3

#Prot

0

2000

4000

6000

8000

10000

12000

14000

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


Analysis of Membrane Proteome Fractions

Regular

protocol

Rapid Trypsin

protocol

16876647

25124640971 unique peptides

unique spectra

proteins

326256741362

Dr. Andreas Otto – University of Greifswald


Complete Coverage of the TM Domain

ARTQ_BACSU Rapid Trypsin ARTQ_BACSU regular in solution digest

Dr. Andreas Otto – University of Greifswald


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


Rapid Digestion Protocol


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


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.


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.


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.


For more information, contact

Michael Rosenblatt

[email protected]

For Early Access, contact

Gary Kobs, Global Product

Manager

[email protected]

Thank You

mailto:[email protected]

mailto:[email protected]

faster mass spec - worldwide.promega.com

Documents