peptide mapping using lc/ms - agilent.com · presenters tim rice – applications specialist,...

Peptide Mapping Using LC/MS

A step-by-step workflow

review

April 18, 2014

Confidentiality Label

1

Presenters

Tim Rice – Applications Specialist, Biocolumns

Bob Giuffre – Applications Specialist, LC Instrumentation

Dawn Stickle – Applications Specialist, Mass Spectrometry

April 18, 2014


2

Overview

Peptide Maps provide more comprehensive analysis than intact

protein analysis

• Identify proteins and their variants

• Determine post-translational modifications (PTMs) and locations

• Confirm protein sequences

April 18, 2014


3

Getting Started

• Choose your column carefully for best results

- Usually, longer columns are better – good to start with 150 mm

- Longer columns enable higher resolution for more complex peptide

maps

- Use 2.1 mm id for mass spec sensitivity

- Smaller pore sizes– 100Å – 150Å - are ideal for small protein fragments

and peptides

April 18, 2014


4

Getting Started

Instrument

• 1290 Infinity Binary LC System with autosampler offers significant advantages for peptide mapping

- Low delay volume (120 µL)

- Low dead volume

- Excellent pump mixing performance

- 1290 Infinity LC Quaternary pump offers even more automated mixing capabilities with BlendAssist

• Agilent 6530 Q-TOF Mass spec - State-of-the-art mass spectrometry system

- BioConfirm software identifies proteins, PTMs, their locations, and protein sequences

April 18, 2014


5

Getting Started

Mobile Phase

• Acetronitrile and water with a formic acid is ideal

- Avoid TFA when using MS

• Use a low-pH acetonitrile buffer gradient

- Facilitates the separation of a wide range of peptides

- Helps to denature the peptide, improving retention and resolution

- The formic acid increases retention of small poorly retained peptides

April 18, 2014


6

Agilent Solvent Safety Caps

• Ensure that dangerous fumes don’t leach into the lab

environment

• Maintain consistency of solvents

• Easy to install and re-use

April 18, 2014


7

Digesting the Protein

Reagents to have on hand:

- 100 mM ammonium bicarbonate (ABC)

- Trypsin stock

- 200 mM dithiothreitol (DTT)

- 200 mM iodoacetimide (IAM)

• Perform digestion at optimal pH 7.5 – 8.5, 37 °C

April 18, 2014


8

Protein Digestion Step-by-Step

1) Denature protein

- Start with 0.5 mg total protein in a 0.5 mL Eppendorf tube

- Add 25 µL ammonium bicarbonate (ABC) stock solution

- Add 25 µL tetrafluoroethylene (TFE) denaturation agent

- Add 1.0 µL DTT stock solution, and vortex to mix

- Heat 60 °C for 45 minutes to 1 hour then cool to room temperature

April 18, 2014


9

Request a copy of the Peptide Mapping “How to”

Guide: visit www.agilent.com/chem/getbioguides


2) Alkylate

- Add 4.0 µL IAM stock solution

- Vortex briefly

- Incubate sample in a foil-covered rack at room temperature for one

hour

3) Quench excess of IAM

- Add 1.0 µL DTT stock solution to destroy excess IAM

- Wrap in foil; stand for one hour at room temperature

April 18, 2014


10


4) Trypsin Digestion

- Prepare fresh stock of trypsin in 100 µL of 50 mM acetic acid

- Add trypsin stock solution at 1:20 to 1:50 by mass to enzyme: substrate

- Vortex briefly

- Place tube in heater and incubate at 37 °C for 4 – 18 hours

- Cool solution

5) Lower digestion pH to halt trypsin activity

- Add 1.0 µL neat formic acid

- If desalting, use TFA to aid in peptide binding to the resin

- Vortex briefly

- Check pH and adjust as necessary

April 18, 2014


11

Setting up the Analysis

Typical starting conditions for peptide mapping:

- 40 °C is ideal starting temperature

- Linear gradient 3 – 65% of the B bottle

- UV setting: 215 nm and 280 nm

- Run time: 75 min. (starting time – may be optimized later) when using

AdvanceBio Peptide Mapping column

April 18, 2014


12

Optimizing Peptide Mapping Methods

• Elevating temperature produces narrower peaks and may

change selectivity

April 18, 2014


13

30 °C

60 °C

Myoglobin Tryptic Digest

Optimizing Peptide Mapping Methods Adjustments to detection

April 18, 2014


14

min 0 10 20 30 40 50

mAU

0

10

20

30

40

50

min 0 10 20 30 40 50

mAU

-1

0

1

2

3

4

220 nm

280 nm

Optimizing Peptide Mapping Methods

Column length and flow rate

April 18, 2014


15

Optimizing Peptide Mapping Methods: Speed

Starting Method: 75 minutes

April 18, 2014


16

min 0 10 20 30 40 50 60

mAU

0

20

40

60

80

100

120

140

160

2.1 x 150 mm

Gradient: 10 – 40% B

Flow Rate: 0.2 mL/min.

Gradient, 40 minutes

Time (min) %B

0 3

70 35

72 90

75 90

Optmizing for speed: 40 minute run

April 18, 2014


17

min 0 5 10 15 20 25 30 35

mAU

0

20

40

60

80

100

120

2.1 x 150 mm




Time (min) %B

0 3

35 35

37 90

40 90

Going even faster: 14 minute run

April 18, 2014


18

min 0 2 4 6 8 10 12

mAU

0

20

40

60

80

100

120

140

160 2.1 x 100 mm




Time (min) %B

0 3

10 35

12 90

414 90

Comparison of three runs

April 18, 2014


19

2.1 x 150 mm, 75 min. run

min 0 5 10 15 20 25 30 35

mAU

0

20

40

60

80

100

120

2.1 x 150 mm, 40 min. run

min 0 10 20 30 40 50 60

mAU

0

20

40

60

80

100

120

140

160

min 0 2 4 6 8 10 12

mAU

0

20

40

60

80

100

120

140

160

2.1 x 100 mm, 14 min. run

Verifying Data Accuracy: LC/MS

LC/MS data significantly enhances the information content

available from peptide mapping

- High sequence coverage is an indicator that an optimal

separation has been achieved.

April 18, 2014


20

Total Ion Chromatograms

April 18, 2014


21

TIC: AdvanceBio Peptide Mapping column, 14 min. Analysis, 0.6mL/min, 433 Bar

TIC: AdvanceBio Peptide Mapping column, 40 min. Analysis, 0.2mL/min, 140 Bar

8 x10

0

0.75

1.75

2.75

3.75

Acquisition Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

inte

nsity

8 x10

0

0.6

1.6

2.6

3.6

4.6

Acquisition Time (min) 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13

inte

nsity

Sequence Coverage Comparison

April 18, 2014


22

14 min. – 99.63% sequence coverage

40 min.-- 99.84% sequence coverage

Rapid PTM Profiling: Deamidation

April 18, 2014


23

Native peptide

Deamidated

form 2

Deamidated

form 1

Native peptide

Deamidated

form 2

Deamidated

form 1

40 min ECC 14 min ECC

AdvanceBio Peptide Mapping Columns

• Performance (efficiency, resolution) similar to sub-2 µm

columns, without the high backpressure

April 18, 2014


24

Comparison of sub-2 µm UHPLC column to AdvanceBio Peptide Mapping columns

April 18, 2014


25

TIC: AdvanceBio Peptide Mapping

column

2.1 x 100mm

TIC: UHPLC (not Agilent): 2.1

x 100mm

inte

nsity

8 x10

0

0.6

1.6

2.6

3.6

4.6

Acquisition Time (min) 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13

14 min. Analysis, 0.6mL/min,

433 Bar

14 min. Analysis, 0.6mL/min, 700 Bar 8 x108

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Acquisition Time (min)

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13

inte

nsity

ECC Comparison

April 18, 2014


26

99% sequence

coverage

99.63%

sequence

coverage

UHPLC Column (not Agilent)

2.1 x 100 mm

Agilent AdvanceBio Peptide Mapping

2.1 x 100 mm

In closing

• Agilent has resources to help you get great peptide mapping results

• Look for biopharma guides, including the Peptide Mapping “How to” guide, at www.agilent.com/chem/getbioguides

• Check out our LC/MS publication warehouse – “Inside LCMS” at www.agilent.com/chem/insidelcms

• Look for the Agilent peptide mapping standard to help you ensure optimal system performance – p/n 5190-0583

Thank you!

April 18, 2014


27

http://www.agilent.com/chem/insidelcms

peptide mapping using lc/ms - agilent.com · presenters tim rice – applications specialist,...

Documents