IntroductionPeptide mapping is the common analytical technique for characterizing biologic therapeutic proteins. The protein is fi rst digested into smaller peptides enzymatically using one or two proteases. The peptide mixture is typically separated by reversed-phase chromatography and detected by UV or mass spectrometry. Mass spectrometry provides the masses of the peptides, which greatly enhances the information content for peptide mapping. With the additional dimension of separation by mass, coeluting peptides can be easily identifi ed and the gradient length signifi cantly reduced. In addition, tandem mass spectrometry can fragment the peptides into smaller pieces and provide evidence for the amino acid sequence of the peptide and the post-translational modifi cations (PTM) such as glycosylation, phosphorylation, oxidation, and deamidation.

This Application Note describes how peptide mapping of an IgG (1) therapeutic monoclonal antibody (mAb) was performed using a novel superfi cially porous peptide mapping column coupled to an Agilent 6550 iFunnel Q-TOF LC/MS System. The unique resolving power of the peptide column to maximize resolution and effi ciency in combination with the MS/MS analysis ensured confi dent and sensitive identifi cation of peptides and resulted in high sequence coverage during a rapid runtime of 15 minutes. The 6550 iFunnel Q-TOF LC/MS System delivered excellent mass accuracy and sensitivity for peptide identifi cation, and the MS/MS capability helped to confi rm modifi ed peptide identifi cations with high confi dence. Additionally, optimization of the IgG (1) peptide gradient and LC/MS/MS parameters were explored for achieving robust, rapid, and reliable mAb peptide maps.

High Resolution and Rapid Peptide Mapping of Monoclonal Antibody Using an Agilent 1290 Infi nity UHPLC and an Agilent 6550 iFunnel Q-TOF LC/MS System

Application Note

AuthorsNing TangAgilent Technologies, Inc.Santa Clara, CA, USA

James Martosella and Alex ZhuAgilent Technologies, Inc.Wilmington, DE, USA

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ExperimentalSample preparationTwenty microliters of mAb IgG1 (30 mg/mL) was fi rst mixed with 20 µL of 100 mM ammonium bicarbonate (pH 8). Then, 50 µL of trifl uorolethanol (TFE) and 5 µL of 200 mM dithiothreitol (DTT) were added to the protein sample and heated at 65 °C for 30 minutes to denature and reduce the protein. After the protein was cooled to room temperature, 20 µL of 200 mM iodoacetamide (IAM) was added to the sample. The sample was kept at room temperature in the dark for 1 hour. Subsequently, 5 µL of DTT was added in the sample to react with the excess IAM for 1 hour. The sample was diluted with 200 µL of 100 mM ammonium bicarbonate and 600 µL of water. 40 µL of trypsin was added to the sample and incubated at 37 °C with shaking (300 rpm) overnight. After overnight incubation, 5 µL of neat formic acid was added to quench the digest.

InstrumentationLC systemAgilent 1290 Infi nity LC System including:

• Agilent 1290 Infi nity Binary Pump G4220A

• Agilent 1290 Infi nity TCC G1316C

• Agilent 1290 Infi nity Sampler G4226A

• Agilent 1290 Infi nity FC/ALS Therm G1330B

MS systemAgilent 6550 iFunnel Q-TOF LC/MS System with Agilent JetStream

Parameter Agilent 1290 Infi nity LC SystemColumn Agilent AdvanceBio Peptide Mapping Column

2.1 × 250 mm, 2.7 µm (p/n 651750-902)Column temperature 60 °CSample thermostat 5 °CMobile phase A 0.1 % formic acid in water Mobile phase B 90 % acetonitrile in water with 0.1 % formic acidGradient 0 minutes 3 % B

10 minutes 35 % B12 minutes 90 % B14 minutes 90 % B15 minutes 3 % B

Stop time 15 minutesPost time 10 minutesFlow rate 0.5 mL/min

LC/MS Parameters

Parameter Agilent 6550 Q-TOF LC/MS SystemIon mode Positive ion mode, Source Agilent Dual JetStreamDrying gas temperature 250 °CDrying gas fl ow 14 L/min Sheath gas temperature 250 °CSheath gas fl ow 11 L/minNebulizer 35 psiCapillary voltage 3,500 VNozzle 0 VMS range (m/z) 300–1,700MS/MS range (m/z) 50–1,700MS scan rate (spectra/second) 8MS/MS scan rate (spectra/second) 3Ramped collision energy Charge state Slope Offset

2 3.1 13 and > 3 3.6 –4.81 3.5 6

Data analysis The data obtained from LC/MS were analyzed using Agilent MassHunter Qualitative Analysis Software B.06 and Agilent MassHunter BioConfi rm Software B.06

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Employing a 60-minute gradient, 95.1 % sequence coverage was achieved with 82 peptides identifi ed by MS/MS. No additional PTMs were identifi ed with the 60-minute gradient. Using the Agilent AdvanceBio Peptide Mapping column and a steep 15-minute gradient, a high resolution and high peak capacity peptide map was achieved with high sequence coverage. Figure 2 shows the overlaid extracted compound chromatograms for (A) 15-minute gradient and (B) 60-minute gradient.

porous peptide mapping column coupled to a 6550 iFunnel Q-TOF LC/MS System. The unique resolving power of the peptide column to maximize resolution and effi ciency, in combination with the MS/MS analysis, ensured confi dent and sensitive identifi cation of peptides.

For mAb IgG1, sequence coverage of 92.1 % was achieved using the 15 minutes gradient. A total of 77 peptides including various PTMs, such as glycosylation, oxidation, and deamidation were identifi ed by MS/MS.

Results and DiscussionA peptide mapping experiment may contain hundreds of peptides with various modifi cations. Automatic data processing improves the effi ciency for data mining. The peptide mapping workfl ow provided by Agilent MassHunter BioConfi rm is depicted in Figure 1. After data acquisition, compounds were extracted automatically based on the chromatography characteristics. The table of compounds was then matched to the list of theoretical peptide digests with potential modifi cation groups. The MS/MS spectrum of each peptide was interpreted by the b/y fragments of the peptides. After the peptides were identifi ed, a sequence coverage map was compiled and the peptide mapping report was automatically generated at the end of the workfl ow.

Traditional peptide mapping methods typically use 90-minute gradients or longer to achieve the desired resolution. With the advancement in UHPLC and related column technologies, gradient time for peptide mapping has been greatly reduced, while coupling LC to MS detection offers an additional dimension of separation in the mass domain. In this work, peptide mapping of an IgG (1) therapeutic monoclonal antibody (mAb) was performed using a novel superfi cially

Acquire data Extract compounds

Match proteinsequences and identify PTMs

Sequencecoverage

Report generation

Acquisition time (min) Acquisition time (min)

Cou

nts

Cou

nts

Peptide mapping workflow

Figure 1. Agilent MassHunter BioConfi rm offers automated peptide mapping data acquisition and analysis workfl ow.

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Deamidation is an important PTM to monitor in peptide mapping. Deamidation can occur during storage, purifi cation, and sample manipulation. Deamidation can be identifi ed by an LC/MS/MS peptide mapping experiment. By adding a negative charge, deamidation can cause structural and functional changes. In the rapid peptide map generated in 15 minutes, light chain peptide 48-63, which contains Asn 53, was identifi ed. Its native form and two deamidated forms, isoaspartic acid and aspartic acid, were separated into three peaks (Figure 3A). The precursor MS of the three peaks are shown in Figure 3B, which clearly shows the addition of 0.98 Da of peaks 2 and 3. Isoaspartic acid and aspartic acid are isobaric. The MS/MS spectra of the three peptides are shown in Figure 3C, wherein, the y-series fragments are the predominant ions.

Figure 2. Identifi ed peptide chromatograms with peptides identifi ed for (A) 15-minute run and (B) 60-minute run.

×106

×106

00.5

11.5

22.5

33.5

44.5

55.5

66.5

77.5

8

B(2

52-2

70)

B(3

23-3

30)

A(4

8-63

)

A(1

09-1

16)

B(4

5-65

)

B(6

8-74

)

B(1

18-1

29)

B(1

30-1

43)

A(1

96-2

10)

B(9

9-11

7)

A(1

56-1

62)

B(2

71-2

84)

A(1

63-1

72)

B(2

98-3

13)

B(3

89-4

05)

B(4

36-4

42)

A(1

8-47

)

B(2

10-2

14)

B(2

19-2

44)

B(3

19-3

22)

A(6

4-68

)

B(2

45-2

51)

B(2

19-2

44)

B(2

45-2

51)

B(3

19-3

22)

B(2

45-2

51)

B(9

9-11

7)

B(9

9-12

9)

A(6

4-68

)

B(3

37-3

51)

A(1

09-1

16)

B(3

57-3

66)

Acquisition time (min)

Acquisition time (min)

Cou

nts

Cou

nts

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 13.5 14 14.5

A

00.40.81.21.62.02.42.83.23.64.04.44.8

B(2

52-2

70)

A(4

8-63

)

B(3

23-3

30)

B(6

8-74

)

B(3

57-3

66)

B(4

5-65

)

A(1

09-1

16)

B(2

45-2

51)

A(1

56-1

62)

A(1

96-2

10)

B(1

30-1

43)

B(1

18-1

29)

B(2

98-3

13)

B(3

89-4

05)

B(2

10-2

14)

A(1

78-1

92)

B(4

36-4

42)

A(2

11-2

18)

B(2

98-3

13)

B(2

19-2

44)

A(4

8-63

)

B(4

13-4

35)

A(6

9-10

8)

B(2

45-2

51)

B(3

67-3

88)

B(7

7-98

)

B(1

44-2

06)

B(3

89-4

05)

B(2

98-3

16)

B(2

89-2

97)

B(2

45-2

51)

B(2

71-2

84)

A(6

4-68

)

B(2

45-2

51)

B(7

5-98

)

B(3

23-3

34)

B(3

52-3

56)

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58

B

×106

00.250.5

0.751

1.251.5

1.752

2.252.5

2.753

3.253.5

3.754

4.254.5

4.755

5.255.5

5.75

A(48

-63)

A(48

-63)

A(48

-63)

Acquisition time (min)

Coun

ts

5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6

Figure 3A. Overlaid extracted compound chromatograms of regular peptide and deamindated peptides.

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All three peptides have the same y10 fragment, however from the y11 fragment and beyond, the ions are 0.98 Da higher for peaks 2 and 3, thus the corresponding sequences for the three peaks are labeled in Figure 3C. Automatic compound extraction (Molecular Feature Extraction) also adds all the ion intensities (isotopes, charge states, adducts, and so forth) belonging to the peptide together (labeled as Volume in the peptide table, Figure 3D). The percentage of modifi ed peptides can easily be derived from the data. In this sample, the two deamidated forms were 3.7 % and 13 % respectively.

Figure 3B. MS spectra of three peptides showing delta mass of 0.98 Da.

00.20.40.60.8

1 * 586.6531

587.3201

587.6537 588.3230

00.5

11.5

2* 586.9787

587.6476

587.9820586.6519 588.6514585.7555585.2533

00.5

11.5

586.9778

587.6471

587.9813 588.6503

Mass-to-charge (m/z)

Coun

ts (%

)Co

unts

(%)

Coun

ts (%

)

585 585.5 586 586.5 587 587.5 588 588.5 589

×102

×101

×101

Figure 3C. MS/MS spectra of three peptides.

×103

×103

×103

0

0.5

1

1.5

2

2.53

227.1755b

2

b2

b2

b3

b3

b3

709.8431 766.3856628.3125

586.6502340.2608511.2581

y14

3+822.9272

277.1549387.1989 473.5691

0

0.25

0.5

0.75

1

1.25

1.5

249.1592

447.2619

390.2353

0

0.5

1

1.5

2586.9786

249.1630 475.3254

390.2355

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880

599.8006

600.2981

600.2898

511.5849

511.5826586.9768

542.7818

542.7799

542.7781

340.2605

340.2612

227.1761

227.1741

628.8038

628.8048710.3377

710.3353

766.8799

766.8816

823.4211

823.4254

N G Y I L

D G Y I L

D G Y I L

Mass-to-charge (m/z)

Cou

nts

Cou

nts

Cou

nts

y10

2+

y10

2+

y10

2+

y13

2+

y13

2+

y13

2+

y14

2+

y14

2+

y14

2+

y15

2+

y15

2+

y15

2+

y11

2+

y11

2+

y11

2+

y12

2+

y12

2+

y12

,b12

-NH3

2+

y14

3+

y14

3+

Figure 3D. Peptide table that showed the native peptide A(48-63) and two deamindated forms with their sequence, modifi cation, retention time, mass and volume.

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This information is subject to change without notice.

© Agilent Technologies, Inc., 2013Published in the USA, November 22, 20135991-3600EN

ConclusionsPeptide mapping of a monoclonal antibody using an Agilent 1290 Infi nity LC System coupled to an Agilent 6550 iFunnel Q-TOF LC/MS System has been demonstrated. LC/MS/MS ensured confi dent and sensitive identifi cation of peptides.

• Rapid and effi cient HPLC peptide mapping of a monoclonal antibody (IgG1) was demonstrated using an Agilent AdvanceBio Peptide Mapping Column. The separation speed and effi ciency are comparable to UHPLC sub-2 µm performance but at a fraction of the system back pressure.

• The 1290 Infi nity LC System provided fast and superior separation power, and 6550 iFunnel Q-TOF LC/MS System delivered excellent mass accuracy and sensitivity for peptide identifi cation. MS/MS capability helped confi rm the peptide ID, especially for modifi ed peptide. A 92 % sequence coverage was achieved with a rapid 15-minute run.

• Agilent MassHunter BioConfi rm software provided automated data extraction, sequence matching, PTM identifi cation, and sequence coverage calculation.