An Integrated Workflow for Peptide
Mapping of Monoclonal Antibodies

Application Note
Biotherapeutics and Biosimilars

Authors

Introduction

David L. Wong

Monoclonal antibodies (mAbs) represent one of the fastest growing classes of drugs
in the pharmaceutical industry. As a protein drug, the manufacturing of an mAb frominitial expression to commercialization is a highly complex process, requiring carefulcharacterization of the mAb in each step. Peptide mapping, among the many analyticalmethods used for mAb characterization, is an essential technique that provides primary
sequence confirmation of the mAb as well as the identification and quantitationof post‑translational modifications (PTMs) such as deamidation, oxidation, andglycosylation1.

Agilent Technologies, Inc.
Santa Clara, CA, USA
Jing Chen
Agilent Technologies, Inc.
Madison, WI, USA

Peptide mapping of an mAb using an LC/MS/MS approach involves the enzymaticcleavage of a purified mAb into peptides, tandem mass spectrometry analysis, anddata interpretation. Sample preparation typically involves multiple steps including
denaturation, reduction, alkylation, and enzymatic digestion. Manual sample preparation
for peptide mapping can be labor-intensive, and it is susceptible to limited scalability andreproducibility.
This application note presents a high-throughput workflow that enables simultaneous,
highly reproducible sample digestion and sample cleanup. This was accomplished using
an Agilent AssayMAP Bravo liquid-handling robot, sensitive and accurate spectrum
acquisition with an Agilent 1290 Infinity II LC system coupled to an Agilent 6550 Q-TOF,
and automated data analysis using Agilent MassHunter BioConfirm software.

Experimental
Materials

Monoclonal antibody standard RM 8671,
was purchased from National Instituteof Standard & Technology (NIST). Mass
spec grade Trypsin/Lys-C enzyme
mix was obtained from Promega. Allother chemicals were purchased from
Sigma‑Aldrich. AssayMAP C18 cartridges
were from Agilent Technologies.

Sample preparation

NIST mAb standard RM 8671, wasdenatured, reduced, alkylated, digested,and desalted using the AssayMAP
Bravo. After diluting samples to 10, 5, 1,
and 0.5 µg/µL in DI water (8 replicatesfor each concentration), 10 µL of eachsample was transferred to a 96-well,opaque, U-bottom plate. Sample
preparation was orchestrated by Protein
Sample Prep Workbench software. The
In-Solution Digestion module was used
to perform sample digestion: 30 µL of
denaturing and reducing reagent (8 M
guanidine, 13.3 mM TCEP, pH 7.5) was
added to each sample. The plate wassealed and incubated off deck at 37 °C for
1 hour.
After unsealing the plate, the In-Solution
digestion application was used to transfer
10 μL of alkylating agent (132 mM
iodoacetamide) from a lidded opaque
96-well U-bottom plate to each sample.
The samples were mixed. The sample
plate was lidded, then incubated in thedark at room temperature for 30 minutes.

Then, 210 μL of diluent (50 mM Tris,
pH 7.5) was added to the samples,
followed by 5 μL of Typsin/LysC Comboof various concentrations (1:40 enzyme toprotein) diluted in 50 mM acetic acid.
The samples were sealed and incubatedat 37 °C for 2 hours off deck. An
additional 5 µL of Typsin/LysC Combo
was added to the samples, and thesamples were incubated off deck at 37 °C
for an additional 14 hours. The next day,
30 µL of 10 % TFA was transferred tothe sample plate to acidify the samples
using the AssayMAP reagent transfer
utility. The Peptide Cleanup application
was used to desalt the samples using C18
cartridges. The cartridges were primed
with 100 µL of 60 % ACN, 0.1 % TFA,
equilibrated with 50 µL of 0.1 % TFA,
loaded with digested samples at a flowrate of 5 µL/min, washed with 50 µL
of 0.1 % TFA, and eluted with 15 µL
of 60 % ACN, 0.1 % TFA at a flow rateof 3 µL/min into 165 µL of 0.1 % FA
to reduce the ACN concentration to
5 %. Then, 0.5 µg of each sample wereanalyzed by LC/MS.

LC/MS Analysis
Instrumentation
LC system
Agilent 1290 Infinity II LC System
including:•

Agilent 1290 Infinity II High Speed
Pump G7120A•

Agilent 1290 Infinity II Multisampler
G7167B•

Agilent 1290 Infinity II Thermostatted
Column Compartment G7116B

2

MS system
Agilent 6550 iFunnel Q-TOF LC/MS
System with Dual Agilent JetStream
LC/MS/MS and LC/MS analyses wereconducted on an Agilent 1290 Infinity II
LC system coupled to an Agilent 6550
iFunnel Q-TOF LC/MS system equippedwith a Dual JetStream ESI source.
LC separation was obtained with an
Agilent AdvanceBio Peptide Mappingcolumn (2.1 × 150 mm, 2.7 µm). Tables 1
and 2 summarize the LC/MS/MS
parameters used. For LC/MS analyses,
the same LC gradient and source
parameters were used. MS data wereacquired at five spectra per second.

Data analysis

Raw data acquired from LC/MS/MS were
processed using Agilent MassHunter
BioConfirm B.08 software. In brief, raw
data were first processed using the
Molecular Feature Extractor (MFE).
The Find Peptide parameters were
set to default, except that the m/z
range was set to not restricted, andfor peak filters, more than 500 counts
was set for peak heights. The results
were searched against the NIST mAb
sequence with fixed modifications foralkylation (C) and variable modifications
for oxidation (M), deamidation (NQ),
pyroglutamate (Q), and various common
forms of glycosylation (N). The MSmatching tolerance was set to ±10 ppm,
and the MS/MS matching tolerance was
set to ±20 ppm. Two enzymatic missedcleavages were allowed. All identified
peptides contained at least 1 MS/MS
spectrum. Peptides with low BioScores
were manually inspected for validity.

Table 1. Liquid chromatography parameters.
Parameter

Value

ColumnAgilent AdvanceBio Peptide Mapping, 2.1 × 150 mm, 2.7 µm

Thermostat

4 °C

Solvent A

0.1 % Formic acid in water

Solvent B

0.1% Formic acid in acetonitrile

Gradient

0–15 minutes, 5–40 %B
15–18 minutes, 40–90 %B
18–20 minutes, 90 %B

Column temperature

60 °C

Flow rate

0.4 mL/min

Table 2. Mass spectrometer parameters.
Parameter

Value

Gas temperature

290 °C

Drying gas

13 L/min

Nebulizer

35 psig

Sheath gas temperature

275 °C

Sheath gas flow

12 L/min

VCap

4,000 V

Nozzle voltage

2,000 V

Fragmentor

175 V

Quad AMU

100

Acquisition mode

Extended Dynamic Range (2 GHz)

Mass range

m/z 100–1,700

Acquisition rate

5 spectra/sec

Auto MS/MS range

m/z 50–1,700

Minimum MS/MS acquisition rate

3 spectra/sec

Isolation width

Medium (~ 4 m/z)

Precursors/cycle

Top 10Collision energy

3.6*(m/z)/100 – 4.8

Threshold for MS/MS

1,000 counts and 0.001 %Dynamic exclusion

On; 3 repeat then exclude for 0.2 minutes

Precursor abundance based scan speed

Yes

Target25,000Use MS/MS accumulation time limit

Yes

Purity100 % stringency, 30 % cutoff

Isotope model

PeptidesSort precursors

By abundance only; +2, +3, >+3

3

Results and Discussion
Integrated workflow using
Agilent AssayMAP Bravo,
Agilent 6550 Q-TOF LC/MS/MS,
and Agilent BioConfirm B.08

necessary to compare different batches
across months or years. In addition,
manual sample preparation can bedifficult to scale up for high-throughput
applications such as clone selection.

Although peptide mapping is routinely
performed for biopharmaceutical mAb
characterization, the sample preparationis complex and essential to the qualityof results. Analytical variance may
result from subtle differences among
techniques, technicians, or different
partner labs. This variation can beespecially challenging when it is

Samplepreparation

The Agilent AssayMAP Bravo automates
the physical work of sample preparation
for peptide mapping, it accelerates theprocess, and improves the reproducibility.
The AssayMAP software provides
easy‑to‑use applications specificallydesigned to address various types ofprotein sample prep workflows, including
In‑Solution Digestion and Peptide
Cleanup. The software directs the robot to

LC/MS/MSexecute essential steps such as sample
denaturation, reduction, alkylation,
enzymatic digestion, and sample cleanup
for LC/MS analysis. This open accessautomation solution is specificallydesigned for protein sample preparation,and the automation of these mechanicalsteps (Figures 1 and 2) allows the analyst
to walk away to perform other tasks while
the samples are being prepared. Sample
prep methods are also transferable
among AssayMAP systems in different
labs to extend reproducibility benefits
across an organization.

Data analysis

Figure 1. Integrated workflow for peptide mapping mAbs, from automated sample preparation on the
Agilent AssayMAP Bravo Platform to Agilent 6550 Q-TOF LC/MS/MS analysis, and Agilent BioConfirm B.08.

Figure 2. The Agilent AssayMAP Peptide Sample Prep Workflow digests, cleans, and optionally fractionates samples for peptide mappingand other workflows.

4

9.5

10.5

B(151-213)

the user-specified mAb sequence, anda sequence coverage map is generated.
Figure 3 illustrates the extracted
compound chromatogram (ECC) of the
NIST mAb, which has been digested with
Trypsin/Lys-C mix. All identified peptides
are labeled with their corresponding
sequence numbers in NIST mAb lightchain and heavy chain.

12.5

B(100-136)

B(6-40)

11.5

A(61-102)

B(46-59)
A(53-102)

A(126-41)
A(108-125)

B(324-337)

7.5
6.5
8.5
Acquisition time (min)A(126-141)

B(390-406)

B(390-406)

A(107-125)

B(226-251)

B(364-373)

B(137-150)

B(278-291)
A(169-182)
B(364-373)

A(29-38)

5.5

B(414-436)

B(437-443)

B(1-5)
A(29-44)4.5

B(305-320)

3.5

B(334-363)

B(78-83)
B(252-258)

2.5

B(412-436)

A(45-52)

A(190-206)

B(259-277)

B(305-320)

of a digested mAb sample contains
numerous peptides, of which many
may have various PTMs, so samples
are complex. Once the MS data are
acquired, the BioConfirm peptide mapping
workflow can be used to perform theMFE processing, and generate a listof compounds (that is, peptides) fromthe highly complex data. The list ofcompounds is then matched against

B(330-337)

1.5

A(149-168)

B(407-411)
B(60-66)
A(53-60)
B(67-73)
B(78-83)

A(207-213)
A(103-106)

0.5

B(60-68)

B(217-221)

×107
1.60
1.55
1.50
1.45
1.40
1.35
1.30
1.25
1.20
1.15
1.10
1.05
1.00
0.95
0.90
0.85
0.80
0.75
0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0

A(207-210)

CountsThe Agilent 6550 iFunnel Q-TOF
LC/MS/MS system has been provento deliver excellent mass accuracy andhighly sensitive MS results for protein
identification and detailed peptide
mapping2. The BioConfirm software
can provide automatic data processingin the areas of MFE, peptide sequence
matching, and protein sequence-coverage
mapping. Typically, an LC/MS/MS run

13.5

14.5

Figure 3. ECC of peptides from Trypsin/Lys-C digested NIST mAb standard RM 8671, separated by Agilent AdvanceBio Peptide
Mapping column. Light- and heavy-chain peptides are marked A and B, respectively.

5

•
•

The presence of b and y fragment
ions
Immonium ions•

Mass accuracy•

MS/MS peak intensity•

Other parameters

Integrating high‑quality MS and MS/MS
data improves the reliability of peptide
matching, and provides a wealth of
information about peptide modifications(Figures 5 and 6).

×10

4

0.9

y13

y11

y9

y7

y5

y3

b2

b4

b6

b8

b10

b12

y1

DSTYSLSSTLTLSKALight chain (169-182)
y8
836.4701

b3-H2O
286.10200.8

y101036.5853

0.7
0.6Counts

To identify peptides with high confidence,
all matched peptides were required
to have <10 ppm MS mass error, andhave at least one confirmatory MS/MS
spectrum. Figure 4 shows examples of the
MS/MS spectra for two selected peptide
fragment ions. The BioConfirm scoringalgorithm considers factors such as:Y
136.0748

0.5
0.4
0.3

T,Y1
74.0594b3
304.1128

Y2
234.1439y4448.2751

0.2
0.1
0

100

200

300

400

500
600
700
Mass-to-charge (m/z)

y9

y7

800

900

y3

y1

y5

1,000

1,100

1,200

ALEWLADIW W DDK

B

b2×103 Heavy chain (46-58)

4.5

y11
1199.6461

b5-H2O
536.1968

y11

5.0

y9
949.5541

y12-H2O2+
y7
641.8463 749.4390

b4

b6

b8

b10

b12

y8
1,048.4728

b2
185.1278

4.0
3.5
2.5
2.0
1.5

y4
y5
b4 563.2438 749.3246
y3 500.2506
b5
377.1650
613.3328

T,l86.0962

0.5
100

200

y7
977.4344

y6
862.4061y2
262.1396

1.00y9
1,161.5557

b3
314.1703

3.0

300

400

500

y10
1,347.6319
y11
1,476.6736

600 700 800 900 1,000 1,100 1,200 1,300 1,400
Mass-to-charge (m/z)

Figure 4. Representative MS/MS spectra of peptides from digested NIST mAb. A) Light chain peptide.
B) Heavy chain peptide.

6

×103

Counts8y27 y25 y23 y21 y19

DIQMTQSPSTLSASVGDRVTITCSASSR

A

b2

b4 b6 b8 b10b3
357.1778

6
4

b4
b5
488.2148 589.2666

Q
2 101.0710
0

100

300

200

Counts

×103
3.0
2.5
2.0
1.5
Q
1.0
101.0714
0.5
0
100

400

500

y27 y25 y23 y21 y19

600
700
800
Mass-to-charge (m/z)

900

DIQMTQSPSTLSASVGDRVTITCSASSR
b2

b3
357.1761b2
229.1176
200

300

b4
504.2107

400

500

b5
605.2618

1,000

1,100

1,200

1,300

991.1526 2+ y22
y21 1120.0530
y263+
y242+
1,076.5369
915.1044
1234.6001
y232+
y252+
1,184.0839
1,308.1205
2+

b4 b6 b8 b10

b7
820.3516

600
700
800
Mass-to-charge (m/z)

×106

7

b7
b6
804.3583
717.3225

y222+ y232+
1,120.0557 1,184.0860
y242+
y212+
1,234.6064
1,076.5399
y252+
y263+
1,300.1281
909.7745

900

1,000

1,100

1,200

1,300

DIQ MTQSPSTLSASVGDRVTITCSASSR(Native, 99.57%)

B

6
5
4
3
2

DIQ MTQSPSTLSASVGDRVTITCSASSR

1(Oxidized, 0.43%)

0
3.4

3.6

3.8

4.0

4.2

4.4

5.0
4.6
4.8
Acquisition time (min)

5.2

5.4

5.6

5.8

6.0

6.2

Figure 5. A) Post-translational modification (methionine oxidation) analysis. MS/MS spectra of native and Met‑oxidized
peptides (Light chain peptide 1-28). Top: native peptide, Bottom: oxidation at Met 4 (green boxes). B) Overlaid ECCs andrelative quantitation analysis of native (blue) and oxidized (red) peptides (light chain peptide 1-28).

7

y2
272.1701

A

×106

y9

y7

b2

b4

b6

b8

b10 b12 b14
y6
810.3841 899.4442

b7-H2O584.2983

150
y15

250
350
y13
y11
y9

450
550
y7
y5
y3

b2

b4

b10

b6

150

b8

y2
272.1704

b3-H2O
228.0957

b4-H2O
229.1337

b6-H2O
485.2335b4

317.1457

250

350

b12

317.1541

150

250

y

1

450

y10
y9
1,196.5793 1,295.6480

y8
1,036.5503

y6811.3725

b14

1,050

y3
435.2333

b5-H2O
386.1769

875.4257

350

450

y15

y13

y11

y9

y7

y5

y3

b2

b4

b6

b8

b10

b12

b14

550

650

y11

y12

750
850
950
Mass-to-charge (m/z)

y6
811.3938

1,395.7052 1,482.7341

988.5088650

y5697.3503

1,450

y8
1,037.5400
b10

b9

550

y4
582.3196

1,150 1,250 1,350
y9
y10
1,197.5674 1,296.6359

y7
924.4531

1,050

y7924.4818

750
850
950
Mass-to-charge (m/z)

1,150

1,250

1,350

y9
1,197.6049

y8
1,037.5687

1,050

1,450

y10
1,296.6729

y1

S G T A S V VC L L N N F Y P R

b4

228.1025

y3

650
750
850
950
y1 Mass-to-charge (m/z)

y5b6
503.2444
696.3441b7584.3021 b7
602.3142y2
272.1787

b3-H2Oy5

y5
696.3440

S G T A S V VC L L N N F Y P R

y12

1,482.7830

1,150

1,250

1,350

1,450

SGTASVVCLLNNFYPR(Native, 97.17%)

B

8

×105

7

2.0
1.5
1.0
0.5
0

6

Countsy11b5-H2O
386.1642

Counts

×103
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0

y13

b6-H2O
485.2299

Counts

×103
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0

y15

S G T A S V VC L L N N F Y P RCounts

×103
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

5
4

9.6

9.8

10.0 10.2 10.4 10.6 10.8 11.0

3
2
SGTASVVCLLNNFYPR(IsoAsp-136, 1.21%)

1
0
9.5

10.0

SGTASVVCLLNNFYPR(Asp-136, 1.38%)

SGTASVVCLLNNFYPR(IsoAsp-137, 0.25%)
10.5

11.0
Acquisition time (min)

11.5

12.0

12.5

Figure 6. A) Post-Translational Modification (Deamidation) Analysis. MS/MS spectra of native and deamidated
peptides (Light chain: 126-141). Top: Native Peptide, Middle: Deamidation at Asn 136, Bottom: Deamidation at Asn
137. B) Overlaid ECCs and relative quantitation analysis of native and deamidated peptides (light chain: 126-141).
Inset: zoom in view of IsoAsp-136 and IsoAsp-137.

8

The relationship between mAb protein
sequence coverage and the amount ofsample digested was also investigated.
Table 3 summarizes the protein sequence
coverage for NIST mAb digests preparedat different sample concentrations.
On average, greater than 95 % protein
sequence coverage was achieved for all
of NIST mAb digests analyzed (n = 8) for
each protein amount. Figure 7 shows a
representative peptide mapping result
from BioConfirm B.08 software, withprotein sequence coverage.

Table 3. Sequence coverage summary of enzymatically digested NIST mAb at various sample amounts.
Sampledigested(µg)

NIST mAb Digest (replicate number)
1

2

3

4

5

6

7

8

Average

100

96 %

96 %

96 %

95 %

96 %

96 %

95 %

97 %

96 %

50

96 %

96 %

97 %

96 %

96 %

97 %

96 %

96 %

96 %

10

97 %

97 %

97 %

97 %

97 %

97 %

98 %

98 %

97 %

05

97 %

93 %

97 %

97 %

94 %

94 %

97 %

94 %

95 %

Figure 7. Screenshot of Agilent MassHunter BioConfirm software with representative peptide mapping results and protein sequence coverage.

9

Post-translational modification
analysis

Protein methionine oxidation andasparagine deamidation are two of
the most frequent PTMs that havebiological significance. Methionine(Met) is a sulfur‑containing aminoacid that is particularly susceptible tooxidation by reactive oxygen species.
The characterization of Met oxidation isoften critical during the development ofmAb‑based therapeutics.
In our peptide mapping results, a NISTmAb light chain peptide 1-28, whichcontains Met 4, was identified as
partially oxidized. Figure 5A illustrates
the MS/MS spectra comparison of thenative (precursor at m/z 985.8097, +3)
and Met-oxidized peptides (precursor atm/z 991.1414, +3). The major differences(+16 Da) in the b4–b7 fragment ions
(green box) clearly distinguished thenative species from the modified forms,
and indicate the specific location of themodification. Moreover, the MS data
indicate the relative amounts of each
peptide species, with the oxidized form
representing approximately 0.43 %,and the native form representing
approximately 99.57 % of the peptides(Figure 5B).

Deamidation is another common PTM. In
this case, a spontaneous nonenzymatic
reaction modifies an asparagine or a
glutamine by removing an amide group,and replacing it with a carboxylategroup. Typically, asparagine is converted
to aspartic acid (Asp) or isoaspartic
acid (Iso Asp). The rate of deamidation
depends upon primary sequence, protein
structure, and solution properties such as
pH, temperature, and ionic strength3.
In our analyses, three deamidated formsof the NIST mAb light chain peptide
126‑141 were found with MS/MS spectraat three distinct retention times. Figure 6A
shows the MS/MS spectra of the native
and the deamidated peptides, where they-series fragments are the predominantions in the larger mass range, and theb-series fragments are the predominantions in the lower mass range. The y6–y12
fragment ions (orange boxes) all show thesignature mass shift of 0.98 Da, clearly
indicating the presence of deamidationin this region of the peptides. In addition,
as the y5 ion (red box) remains the same
(middle panel) as in the native form (toppanel), it is evident that the deamidation
for this peptide occurred at the Asn 136,
and not at Asn 137. In contrast, the y5
ion in the bottom panel (purple box)

10

presents 0.98 Da higher than the masses
in the native sequence, confirming thedeamidation at the Asn 137 position inthis population of peptides.
A quantitative analysis of the MS data
reveals considerably more information
about the deamidation dynamics of thislight chain 126-141 peptide. The native,
unmodified form of the peptide madeup 97.17 % of the sample, and three
deamidated variants were observedin different proportions: IsoAsp-136(1.21 %), IsoAsp-137 (0.25 %), andAsp‑136 (1.38 %). Figure 6B summarizes
these results. It is noteworthy that
this kind of position‑specific PTM
information, the relative quantitation ofeach modification, is often critical fortherapeutic drug (mAb) development. This
workflow makes such information readily
accessible. Because peptide‑mapping
workflows are so complex, with so many
sample manipulation steps required to
produce a precisely reduced, alkylated,digested, desalted sample for the LC/MS
measurement, this type of quantitative
analysis benefits greatly from highly
reproducible robotic sample processing,which is easily automated by the
AssayMAP liquid handling system.

Automated peptide mapping
workflow produces highly
reproducible results

eight replicate mAb samples that weredenatured, digested, cleaned up in
separate wells, then injected separately.
For this experiment, all quantitative
measurements were made in MS-onlymode on the 6550 Q-TOF LC/MS/MS
instrument. Twenty-eight peptides with no
missed cleavages were quantified by EIC
for samples prepared with 5 µg of mAb.
Overlaid EICs from two representative
peptides are shown for LC/MS injection
replicates (Figures 8A and 8B) andsample preparation replicates (Figures 8C
and 8D). Of the 28 peptides quantified,

Since this automated workflow solution
makes it easy to scale an experiment up
or down to accommodate any numberof samples, we ran replicate analyses
in this study to evaluate the precisionand reproducibility of the system at
two levels. At one level, we evaluatedthe quantitative reproducibility of the
LC/MS system by injecting the samedigested mAb sample eight times
from one well. Secondly, we analyzed

17 had CVs less than 10 % (average 6 %),seven had CVs between 10 % and 20 %(average 15 %), and four had CVs greaterthan 20 % (Figure 8E). These results are
consistent with others that have beenreported for AssayMAP peptide mapping
studies4.
Although it was not described in thisstudy, the AssayMAP Affinity Purification
application with protein A, protein G, or
streptavidin affinity cartridges allows thisworkflow to be expanded for use withsamples suspended in growth medium,plasma, or other complex matrices.

LC/MS Injection replicates (n = 8)

3
2

×106
3.0

A
LLIYDTSK
LC/MS Replicates
CV: 1.39 %CountsCounts

×106
4

B

2.5

VYACEVTHQGLSSPVTK

2.0

LC/MS Replicates
CV: 1.38 %

1.5
1.0

1

0.5

0

6.0

6.1

6.2

6.3
6.4
Acquisition time (min)

6.5

6.6

0

6.7

5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60
Acquisition time (min)Sample preparation replicates (n = 8)

3
2

C

×106
3.0

D

LLIYDTSK

2.5

VYACEVTHQGLSSPVTK

Sample Prep. Replicates
CV: 3.53 %2.0

Sample Prep. Replicates
CV: 6.33 %CountsCounts

×106
4

1

1.5
1.0
0.5

0

6.1

6.0

6.2

6.3
6.4
Acquisition time (min)

6.5

6.6

6.7

0

5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60
Acquisition time (min)

<10 %

E

10–20 %
>20 %

4

7

17

Figure 8. EICs of representative peptides digested from 5 µg NIST mAb. A, B) LC/MS injection replicates of the same sample (n = 8). C, D)
Complete‑sample-preparation replicates (n = 8). E) Pie chart of the number of peptides with %CV less than 10 %, between 10 % and 20 %, and over 20 %.

11Conclusions
The Agilent peptide mapping workflow
solution integrates state-of-the-art liquidhandling robotics, high-performance
chromatography technologies,
accurate‑mass Q-TOF LC/MS, andpowerful software to automate andstreamline a complex process that is
critical to the development of mAb
biologics. This workflow featured:•

An Agilent AssayMAP Bravo system,
which enabled automated enzymatic
digestion and sample cleanup withexcellent reproducibility.

•

Superior LC resolution from an
Agilent AdvanceBio Peptide Mappingcolumn, which enabled rapid
separation for improved analyticallaboratory efficiency.

•

The highly accurate and sensitive
Agilent 6550 Q-TOF LC/MS/MS
system, which produced unbiased
peptide mapping data to achievecomprehensive sequence coverageof monoclonal antibody proteins.

•

Agilent MassHunter BioConfirm data
analysis software, which performed
automated data extraction, sequence
matching, PTM identification, andsequence coverage calculation.

References
1. Greer, F. M. MS Analysis of
BioPharmaceutical Products.
Innovation in Pharmaceutical
Technology 2001, 83-90.
2. High Resolution and Rapid Peptide
Mapping of Monoclonal Antibody
Using an Agilent 1290 Infinity UHPLC
and an Agilent 6550 iFunnel Q-TOF
LC/MS System, Agilent Technologies,
publication number 5991-3600EN.
3. Robinson, N. E. Protein Deamidation.
PNAS 2002, 99(8), 5283-5288.
4. Automation for LC/MS Sample
Preparation: High Throughput
In-Solution Digestion and
Peptide Cleanup Enabled by the
Agilent AssayMAP Bravo Platform,
Agilent Technologies, publication
number 5991-2957EN.

www.agilent.com/chem/peptidemapping
For Research Use Only. Not for use in diagnostic
procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc., 2017
Published in the USA, February 8, 20175991-7811EN