Bio Columns for Peptide Mapping - · PDF filePeptide Plus Column Anne Blackwell, Ph.D. BioColumns Product Support Scientist June 7, 2017 ASMS 1 ... (SAX, SCX, WAX, WCX) PL SAX PL SCX

CQA Assessment by Peptide Mapping Using LC/MS with an AdvanceBio Peptide Plus Column

Anne Blackwell, Ph.D.

BioColumns Product Support Scientist

June 7, 2017

ASMS

1

For Research Use Only. Not for use in diagnostic procedures.

Analytical groups are tasked with method development, sample analysis, and method transfer

Centrifuge

Biomarker ID

& validation

DNA

Vector

HT Clone

Screening &

Selection

mAb Generation Platform

e.g. transgenic mice Cell Culture Scale-up & Production

Human

gene

Establishment of genetically

engineered cells (e.g. CHO) that

produce desired product

Analytical Groups Develop analytical methods;

analyze samples from other

functions, methods transfer

QA/QC

Samples Samples Samples Samples

Samples Samples

Methods

Market Manufacturing Pre-clinical Clinical Chromatography

Column #1

Chromatography

Column #2

Chromatography

Column #3

2


Pyro-

Glutamate

Deamidation

/Oxidation

Fragmentation

(Hinge)

Glycosylation

(G0, G1, G2)

Truncation

(Lys 0, 1, 2)

Disulfide

Shuffling

Aggregation

HILIC

IEC

IEC

IEC

RP

SEC

RP

LC/MS Intact mass

Fragment mass

Identification

PTM ID & locations

Glycan ID

Critical Quality Attributes & Testing Methods

Not just used for monoclonal antibodies – same techniques

are used for any potentially therapeutic protein

3


Peptide Mapping Workflow Solutions to Accelerate Biomolecule Characterization

4

Delivery of Results

Reporting

Sequence Matching

Determine Post Translational

Modifications

Data Acquisition

Sample Preparation

Chromatographic Separation

Complete Agilent Workflow Solution

B(6

7-7

3)

B ( 6 0 - 6 6 )

A(1

03

-10

6)

B(3

30

-33

7)

B(2

78

-29

1)

Peptide mapping chromatogram Peptide ID by MS/MS


Sample Preparation

Denaturation, Reduction, &

Alkylation

Digestion

Enrichment & Cleanup

Sample Preparation for Peptide Mapping Depletion of matrix proteins,

enrichment of target protein,

dialysis or desalting

Reduce disulfide bonds, and

block to prevent re-formation

Trypsin is the most common

digestion enzyme, but others may

be called for depending on the

protein and experiment.

Sample concentration, enrichment

for specific PTMs, or desalting

A time consuming, labor-intensive process

to do manually, with multiple steps that can

be optimized.

5


AssayMAP Bravo – Sample Prep Automation

6

Peptide mapping

Detect


AssayMAP Sample Prep Portfolio

Fractionation

Antibody

Purification

Immunoaffinity

Purification

Phosphopeptide

Enrichment

N-glycan

Sample Prep

Protein

Digestion

Peptide Clean

Up

Protein Clean

Up

7


General Considerations for Peptide Mapping Column Selection

Instrument capabilities and requirements - UV vs MS detection or both

- Pressure capabilities

Mobile phase requirements - High pH required for sample

- TFA vs Formic acid

Sample - Hydrophilic vs hydrophobic peptides

- Larger polypeptides present

Column dimensions - Generally prefer longer columns, especially for more complicated maps

- 50, 150, and 250 mm lengths available

- Use 2.1 mm i.d. for MS-sensitivity

- 3.0 and 4.6 mm i.d. also available

- Smaller pore sizes ideal, usually 100-150 Å

8


Detectors for Peptide Mapping

UV

• Often used in routine process measurements,

QA/QC labs

• No peptide mass or structural information; relies on

reproducibility of RT, peak area, and peak area

ratios

• Traditionally uses TFA modifier

MS

• Often used in discovery/early development

stage, R&D labs

• MW and structural information (MS/MS) to

assist with co-elution challenges, verify

sequence coverage, and to ID unknown peaks

when they appear

• Generally prefer formic acid modifier to avoid

ion suppression from TFA

9


Mobile Phases for Peptide Mapping

Ion pairing agents used for RP LC

• Increase retention of small, poorly retained peptides

• Block exposed silanol sites on silica-based columns

• Acid helps solubilize and ionize peptide and protein samples

TFA works well on “traditional” silica-

based columns, but formic acid does not

• Formic acid produces broad, asymmetric peaks

• Poor resolution and poor peak capacity

Need column solution for formic acid

modifier

• AdvanceBio Peptide Plus

• Charged surface chemistry solves the problems associated with formic acid

10


Agilent Bio-LC Column Portfolio Agilent Bio-LC Columns

Affinity

Bio-Monolith Protein A

Bio-Monolith Protein G

Multiple Affinity Removal System

Reversed Phase

AdvanceBio Peptide Plus

AdvanceBio Peptide Mapping

AdvanceBio RP mAb

AdvanceBio

Oligonucleotides

AdvanceBio Desalting-RP

PLRP-S

ZORBAX RRHD 300Å, 1.8 µm

AdvanceBio Amino Acid Analysis

ZORBAX Amino Acid Analysis

ZORBAX 300SB

Poroshell 300

HILIC

AdvanceBio Glycan Mapping

ZORBAX RRHD 300-HILIC

Size Exclusion

AdvanceBio SEC

Bio SEC-3

Bio SEC-5

ProSEC 300S

ZORBAX GF-250

ZORBAX GF-450

Ion Exchange

Bio mAb

Bio IEX

(SAX, SCX, WAX, WCX)

PL SAX

PL SCX

Bio-Monolith

(QA, DEAE, SO3)

11


Agilent Bio-LC Column Portfolio

Agilent Bio-LC Reverse Phase Columns for Protein Analysis

Intact Proteins (& Fragments)

PLRP-S, 5 µm, 1000 Å

AdvanceBio RP mAb

ZORBAX RRHD 1.8 µm, 300 Å

ZORBAX 300SB

Poroshell 300

AdvanceBio Desalting-RP

Peptides



ZORBAX Eclipse Plus C18

ZORBAX 300SB*

Amino Acids

AdvanceBio Amino Acid Analysis

ZORBAX Amino Acid Analysis

12

*unusually large pore size for peptide mapping,

but some people really like it nonetheless For Research Use Only. Not for use in diagnostic procedures.


Poroshell particle UHPLC performance at lower pressure

Unique charge hybrid/C18 bonded phase Improved peak shape and peak capacity for peptide

analysis with formic acid and MS detection

To provide AdvanceBio Peptide Plus that gives

benefits in accuracy and reproducibility of results

for characterization of mAb identity, PTMs

13


Columns Individually Tested for Efficiency

14

AdvanceBio Peptide Plus Column


Column Lots QA Tested Specifically for Peptide Separations

15

Peptides Standard

(p/n G2455-85001) AdvanceBio Peptide Plus Column For Research Use Only. Not for use in diagnostic procedures.

LC/MS Instrumentation

16

• 1290 Infinity II LC system

• 6545XT AdvanceBio LC/Q-TOF

• BioConfirm B.08 for intact protein

and peptide mapping data analysis


6545XT Features for Peptide Mapping

• Sensitive peptide detection featuring Agilent Jet Stream

• Quick-start peptide mapping method

• Access low intensity peptides/PTMs with the new

IterativeMS/MS mode

• Sub-ppm mass accuracy with 50k resolution from

improved beam optics

• Peptide fragmentation performance verification at

install

• Ease of maintenance with vent-free capillary removal



Method Conditions

18

Parameter 1290 Infinity II LC

Column AdvanceBio Peptide Plus 2.1 × 150mm, 2.7µm, 120Å

Mobile phase A: 0.1%FA

B: 0.1% FA in ACN

Gradient

Time (min) %B

0 3

1 3

31 40

33 95

34 95

34.1 3

Post time 10 min

Inj vol 1 µL

Sample thermostat 5 °C

Post time 5 min

Column temp 55 °C

Flow rate 0.5 mL/min

Parameter 6545XT AdvanceBio LC/Q-TOF

Ion mode Positive ion mode, dual AJS ESI (profile)

Drying gas temp 325 °C

Drying gas flow 13 L/min

Sheath gas temp 275 °C

Sheath gas flow 12 L/min

Nebulizer 35 psi

Capillary voltage 4000 V

Fragmentor voltage 175 V

Skimmer voltage 65 V

Oct RF Vpp 750 V

Acquisition parameters Data were acquired at 2 GHz Extended Dynamic

Range

MS mass range 100–1700 m/z

MS/MS mass range 50 – 1700

MS scan rate (spectra/second): 8

MS/MS scan rate (spectra/second): 3

Ramped collision energy

Charge state Slope Offset

2 3.1 1

3 and > 3 3.6 –4.8

Data analysis BioConfirm software B.08.08

All results shown are with formic

acid mobile phase


Effect of Flow Rate on Peak Capacity


19

TIC

• Increasing the flow rate from 0.1 mL/min to

0.5 mL/min leads to an increase of twice the

peak capacity

8x10

0

0.5

1

1.5

8x10

0

0.5

1

8x10

0

0.5

1

8x10

0

0.5

1

8x10

0

0.5

1

1.5

8x10

0

0.5

1

1.5

Counts vs. 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 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

0.1 ml/min

0.2 ml/min

0.3 ml/min

0.4 ml/min

0.5 ml/min

0.6 ml/min


Effect of Gradient Length on Peak Capacity


20

• Increasing the gradient time from 0 min to 60 min

leads to an increase 2.5 times peak capacity,

• After 30min, no significant increase in peak capacity

TIC

8x10

0

0.5

1

1.5

8x10

0

0.5

1

8x10

0

0.4

0.8

1.2

8x10

0

0.4

0.8

1.2

8x10

0

0.4

0.8

1.2

8x10

0

0.4

0.8

1.2

Counts vs. 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 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

10 min

20 min

30 min

40 min

50 min

60 min


Effect of Temperature on Peak Capacity


21

LC/MS Chromatogram

• Higher temperature produces narrower peaks, improving resolution

• Change in temperature changes selectivity

• ~20% more peaks resolved by increasing the column temperature to 550C 6x10

0

1

2

6x10

0

1

2

3

Co unts vs. 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 40

45oC

55oC

Pc = 504

Pc = 600


Good Peak Shape & Retention Time Stability with High Mass Load

22

Sample: 3-peptide

mixture in 0.1%FA

• Bradykinin

• Angiotensin II

• Venin substrate

All 2.1 x 50 mm columns

0.6 ug

6 ug

0.6 ug

6 ug

0.6 ug

6 ug


Cortecs Kinetc

0.6 ug

6 ug


Key for transferring

methods between

LC/MS and LC/UV


AdvanceBio Peptide Plus Competitor 1

Competitor 2 Competitor 3

Highly Reproducible Separations

23

n = 10 %RSD

Peak no. RT FWHM Area Height

1 0.20 1.13 0.31 1.36

2 0.06 1.57 5.76 2.54

3 0.06 1.55 4.27 1.39

4 0.06 1.02 4.60 1.23

5 0.07 0.91 3.41 1.53

6 0.05 0.86 2.69 0.74

7 0.06 3.22 5.79 3.33

8 0.04 0.99 3.81 1.12

9 0.02 0.92 2.66 1.31

10 0.02 0.95 4.22 1.80

8x10

0

1

2

3

4

5

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

Cou

nts

Hydrophilic Hydrophobic

• Consistent chromatography

• RSD values demonstrate the robustness of the method

and precision of the LC system

• Especially crucial for LC/UV peptide mapping

1

2

3

4 5

6

7

8

9

10



Lifetime Testing

24

Plates dropped < 10%

Rt dropped < 10%

Retention time %

Plates count %

• The column has subjected to 1000

injections and lost no more than 10%

of the initial plate and retention value

at the end of the test

• Good chemical stability under formic

acid conditions

Column: AdvanceBio Peptide Plus

Sample: 10 peptide standard (5190-0583)

Injection: 3 µL

Flow rate: 0.5 mL/min

Mobile phase: A – 0.1%FA

B – 0.1% FA in ACN

% P

late

count


8x 10

0

0. 5

1

1. 5

2

2. 5

3

3. 5

4

4. 5

5

5. 5

6

8x 10

0

0. 5

1

1. 5

2

2. 5

3

3. 5

4

4. 5

5

5. 5

6

C ounts v s. 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

12

35

4

67

8

9 10AdvanceBio Peptide Mapping


1

2

3 5

4

67 8

9

10

Column Options for Alternate Selectivity

25

10 Peptide Standard (p/n 5190-0583)

TIC

6. Renin substrate porcine

7. [Ace-F-3,-2 H-1] Angiotensinogen (1-14)

8. Ser/Thr Protein Phosphatase (15- 31)

9. [F14] Ser/Thr Protein Phosphatase (15-31)

10. Melittin (honey bee venom)

1. Bradykinin frag 1-7

2. Bradykinin

3. Angiotensin II (human)

4. Neurotensin

5. Angiotensin I (human)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

FWH

M

Peptide peak

AdvBio Peptide Mapping

AdvBio Peptide Plus

Better


mAb Peptide Mapping

26

LC/MS of mAb tryptic digest on 2.1 x 150mm AdvanceBio Peptide Plus Column

Hydrophilic peptide retention

Narrow Peaks with baseline resolution

Hydrophobic peptide elution

Reduced and fast analysis time

Critical and desired peptide mapping

components to achieve fast, selective,

and highly efficient peptide separations

across a wide dynamic range.

8 x10

0

1

2

3

4

5


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

Co

un

ts

Hydrophilic Hydrophobic


LC/MS Peptide Map

27

sequence coverage 99.25% mass accuracy of 5 ppm



BioConfirm for Peptide Mapping


28

MS spectrum MS/MS fragment spectrum

Biomolecules listb and y ion list

Sequence map

Chromatograms


Identification of C-terminal Truncation

29

SLSLSPGK

SLSLSPG


Analysis of Post-Translational Modifications

30

• Post-translational modifications (PTMs) on proteins such as monoclonal antibodies (mAb) are

critical quality attributes (CQA) that define drug efficacy and safety

• PTM containing peptides represent the minority of the tryptic mixture and often masked by more

dominant peptide signals

• Low stoichiometry & heterogeneity of many post-translational modifications make characterization

of modification sites challenging

• Advantage of high mass load tolerance of AdvanceBio Peptide Plus

• Comprehensive characterization of PTMs requires:

• Separation of native and modified peptides (Column)

• Sensitive detection (Mass Spectrometry)

• Robust LC/MS method


Separation of Oxidized Peptides

31

5 x10

0

1

2

3

4

5

6

8


4 5 6 7 8

7

Co

un

ts

Oxidized peptide

DLTMISR peptide

Relative % of Oxidation = 1.69% EIC

2x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

2.75

3

3.25

3.5

3.75

4

4.25

4.5

4.75

5

5.25

5.5

5.75

6

6.25

6.5

6.75

7

7.25

7.5

7.75

8

T74.0604

L,I86.0965

100.8496126.0552

144.0666

171.0775

175.1200

189.0863199.0725

b2217.0827

229.0918

245.1374

y2

262.1507

b3-H2O

312.1557

b3

330.1666

y3

375.2340

409.7209

443.1929

y4

506.2769

575.3696602.3394

y5

619.3600

619.6263

Counts v s. M ass-to-Charge (m/z)

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660

b4-H2Oy2 -NH3

y1

R

3x10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

L,I

86.0960

88.0377

111.0555

144.0642

y1-NH3

158.0920

175.1176

189.0875

b2-H2O

199.0721

b2

217.0824

229.6381

y2

262.1503

286.1798

b3-H2O

312.1563

330.1614

351.6818

y3

375.2351

394.2188

417.2133

426.2170

458.2711

478.2066502.2897

y4

522.2694

571.3618

y5

635.3547

Counts vs. Mass-to-Charge (m/ z)

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660

DLTMISR

MS/MS spectra

Increase in mass (16Da)

Unmodified peptide

Unmodified peptide

Oxidized peptide

DLTMISR



Separation of Deamidated Peptides

32

ASQDVNTAVAWYQQKPGK peptide NTAYLQMNSLR peptide

Relative % of Deamidation = 0.21% – 15.9% Relative % of Deamidation = 9.1%

EIC

NT

AY

LQ

MN

SL

R 1

5.9

%

NT

AY

LQ

MN

SL

R 5

.5%

NT

AY

LQ

MN

SL

R

0.2

3%

NT

AY

LQ

MN

SL

R 0

.97

%

NT

AY

LQ

MN

SL

R

2.5

%

NT

AY

LQ

MN

SL

R

0.2

4%

NT

AY

LQ

MN

SL

R 0

.21

%

NT

AY

LQ

MN

SL

R

Deamidated peptides

non-d

eam

idate

dpepti

de

Deamidated peptide

Non-deamidated peptide

Deamidated peptide


NTAYLQMNSLR

GFYPSDIAVEWESNGQPENNYK

Relative % deamidation = 9.1%


ASQDVNTAVAWYQQKPGK

Deamidated peptide



ASQDVNTAVAWYQQKPGK



33

Traditional Silica AdvanceBio Peptide Plus Sub 2 µm

• Determination of deamidated peptides is an important goal of peptide mapping. Chromatographic resolution is important. MS/MS confirms which peak corresponds to which deamidation site.

• AdvanceBio Peptide Plus enhances analysis because it has higher selectivity for deamidated forms of peptide over the normal

form.

Separation of Deamidated Peptides


VVSVLTVLHQDWLnGK or VVSVLTVLHqDWLNGK m/z 904.9984, doubly charged peptide

Sub 2 µm column requires

much higher back pressure to

achieve similar separation.

Traditional silica

columns don’t

resolve deamidation

as well as charged

surface columns.

Separation of Glycopeptides

34

EIC

G1F = 45.4%

G0F = 40.6%

G2F = 10.1%

G0 = 3.79%

Relative quantification

EEEQYNSTR peptide 3x10

0

0.5

1

1.5

2

3x10

0

1

2

3

4

5x10

0

0.2

0.4

0.6

0.8

1

1.2

5x10

0

0.2

0.4

0.6

0.8

1

4x10

0

0.2

0.4

0.6

0.8

1

1.2

Counts vs. Acquisition Time (min)

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2

EEEQYNSTR

EEEQYNSTR+G0

EEEQYNSTR+G1F

EEEQYNSTR+G0F

EEEQYNSTR+G2F

Non-glycosylated BioConfirm results

Glycosylated



Pyro-

Glutamate

Deamidation

/Oxidation

Fragmentation

(Hinge)

Glycosylation

(G0, G1, G2)

Truncation

(Lys 0, 1, 2)

Disulfide

Shuffling

Aggregation

HILIC

IEC

IEC

IEC

RP

SEC

RP

LC/MS Intact mass

Fragment mass

Identification

PTM ID & locations

Glycan ID

Critical Quality Attributes & Testing Methods

Not just used for monoclonal antibodies – same techniques

are used for any potentially therapeutic protein

With a Multi-Attribute Method, peptide mapping can

monitor many of these CQAs simultaneously.

35


Multi-Attribute Method (MAM)

36

0

5

10

15

20

25

30

35

40

45

50

G1F G0F G2F G0

% m

od

ific

atio

n

RP Heavy chain

HILIC

MAM (Pep Map)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

% m

odifi

catio

n

%

G1F 44.8

G0F 44.7

G2F 8.9

G0 1.5

HILIC RP

Deamidations and Oxidation

monitored by the MAM (Pep Map)

Glycans monitored

Deamidation Oxidation

CEX

Comparison to traditional methods


Choosing a Column Chemistry

For LC/MS work, AdvanceBio Peptide Plus with formic acid is the first choice.

Best peak shape with formic acid, high peak capacity, high mass load tolerance, excellent PTM resolution

(especially deamidation), peak shape and recovery of hydrophobic peptides, unique selectivity

Charged surface chemistry


“Regular” silica chemistry


Eclipse Plus RRHD

ZORBAX 300

vs

37


Exception 1: Sample is especially rich in small,

hydrophilic peptides.

Exception 2: Different selectivity is needed to separate

a key pair.

Poor retention of small, hydrophilic peptides is a feature common to

charged surface columns. In this case, try AdvanceBio Peptide

Mapping or Eclipse Plus C18 first.

Method Development Tips & Reminders

38

• Bonded Phase: C18 is routinely used. Try AdvanceBio Peptide Plus first; for alternate selectivity or highly

hydrophilic samples try AdvanceBio Peptide Mapping or a ZORBAX column.

• Column dimension: Good start with 2.1mm × 150mm, for higher resolution use longer column

• Gradient: Acetonitrile : Water with 0.1% formic acid, other organic solvent substitutions can be used for

different selectivity

Starting gradient time: 0-40% in 30min (1.3%/min)

• Temperature: Higher column temperature can dramatically improve both resolution and recovery (60 °C max)

• Desalt peptide mixture prior to injection

• Sample handling may cause artifacts (peptide modifications)


Troubleshooting – Reasons for < 100% Sequence Coverage

Question to ask: Looking at the protein sequence, what’s missing?

• Very small and/or very hydrophilic peptides?

• If using AdvanceBio Peptide Plus, try AdvanceBio Peptide Mapping for better retention and/or

resolution.

• Very large and/or very hydrophobic peptides?

• Can you go to a higher percent organic mobile phase?

• Are peptides sticking to sample tubes or otherwise lost during sample preparation? Or on the LC?

• If using AdvanceBio Peptide Mapping, try AdvanceBio Peptide Plus

- May even need to consider a C8 column rather than C18

• Very large, or very small peptides of any hydrophobicity?

• Consider a different digestion enzyme


39

More Information

Application Notes:

LC/MS Analysis of Peptide Mapping with

Formic Acid Ion-Pairing Agent

(5991-7574EN)

Enhancing the Quality of Peptide Mapping

Separation for the Analysis of Post-

Translational Modifications

(5991-7875EN)

How-to Guide

40


Standards & Reagents

41

10 Peptide Standard (p/n 5190-0583) HSA peptides Standard

(p/n G2455-85001)

1. Bradykinin frag 1-7

2. Bradykinin

3. Angiotensin II (human)

4. Neurotensin

5. Angiotensin I (human)

6. Renin substrate porcine

7. [Ace-F-3,-2 H-1] Angiotensinogen (1-14)

8. Ser/Thr Protein Phosphatase (15- 31)

9. [F14] Ser/Thr Protein Phosphatase (15-31)

10. Melittin (honey bee venom)

7x 1 0

0

0 . 2

0 . 4

0 . 6

0 . 8

1

1 . 2

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5

Co

un

ts

A cquisition Time (min)

1

23

5 67

10

9

8

4

Blank

1. AAFTE[CAM][CAM]QAADK

2. YLYEIAR

3. LVNEVTEFAK

4. KVPQVSTPTLVEVSR

5. RP[CAM]FSALEVDETYVPK

6. AVMDDFAAFVEK

7. HPYFYAPELLFFAK

Trypsin Digest Methylated BSA Standard

(p/n G1990-85000) Trypsin


Summary

42

MS-compatible AdvanceBio Peptide Plus column delivers high peak capacity with sharp and narrow

peaks using MS-friendly formic acid modifier mobile phase conditions.

AdvanceBio Peptide Plus and AdvanceBio Peptide Mapping are superficially porous columns that yield

low back pressures & STM-like performance, allowing peptide mapping separations to be run on both

HPLC and UHPLC systems

Well-resolved PTM modified peptide peaks enabled reliable mAb peptide maps and development of

Multi-Attribute Methods


Documents

Bio Columns for Peptide Mapping - · PDF filePeptide Plus Column Anne Blackwell, Ph.D. BioColumns Product Support Scientist June 7, 2017 ASMS 1 ... (SAX, SCX, WAX, WCX) PL SAX PL SCX