Top-down N-terminal sequencing of Immunoglobulin subunits with electrospray ionization time of flight mass spectrometry

Analytical Biochemistry 384 (2009) 42–48

Contents lists available at ScienceDirect

Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio

Top-down N-terminal sequencing of Immunoglobulin subunits with electrospray ionization time of flight mass spectrometry Da Ren a,*, Gary D. Pipes a, David Hambly b, Pavel V. Bondarenko a, Michael J. Treuheit b, Himanshu S. Gadgil b,* a b

Formulation and Analytical Resources, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA Analytical and Formulation Sciences, Amgen Inc., 1201 Amgen Court West, Seattle, WA 99119-3105, USA

a r t i c l e

i n f o

Article history: Received 1 May 2008 Available online 21 September 2008 Keywords: IgG Top-down Fragmentation Time of flight

a b s t r a c t An N-terminal top-down sequencing approach was developed for IgG characterization, using high-resolution HPLC separation and collisionally activated dissociation (CAD) on a single-stage LCT Premier time of flight (TOF) mass spectrometer. Fragmentation of the IgG chains on the LCT Premier was optimized by varying the ion guide voltage values. Ion guide 1 voltage had the most significant effect on the fragmentation of the IgG chains. An ion guide 1 voltage value of 100 V was found to be optimum for the N-terminal fragmentation of IgG heavy and light chains, which are approximately 50 and 25 kDa, respectively. The most prominent ion series in this CAD experiment was the terminal b-ion series which allows N-terminal sequencing. Using this technique, we were able to confirm the sequence of up to seven N-terminal residues. Applications of this method for the identification of N-terminal pyroglutamic acid formation will be discussed. The method described could be used as a high-throughput method for the rapid N-terminal sequencing of IgG chains and for the detection of chemical modifications in the terminal residues. Ó 2008 Elsevier Inc. All rights reserved.

The requirements for bio-therapeutic characterization are stringent due to their intended use in patients. A wide array of analytical techniques is often required to characterize bio-therapeutic proteins. Techniques such as N-terminal sequencing and bioassays are used for establishing product identity. Ion exchange chromatography, size exclusion chromatography, capillary electrophoresis, and peptide mapping are often used to determine drug product purity. In recent years, electrospray ionization time of flight mass spectrometry (ESI-TOF MS)1 has become a popular technique for the analysis of therapeutic proteins and IgG molecules [1–4]. TOF-MS allows accurate mass measurements for proteins greater than 150 kDa, with mass errors of less than 20 ppm [2]. Bio-therapeutic proteins are produced with closely controlled procedures, but significant variability still exists in these products, which is often caused by chemical modifications. Most of these modifications cannot be resolved at the intact protein level; i.e., the accurate mass measurement alone is not sufficient for

* Corresponding authors. Fax: +1 805447 3401 (H.S. Gadgil). E-mail addresses: [email protected] (D. Ren), [email protected] (H.S. Gadgil). 1 Abbreviations used: ACN, acetonitrile; CAD, collisionally activated dissociation; ESI-TOF MS, electrospray ionization time of flight mass spectrometry; GdnHCl, guanidine hydrochloride; HC, heavy chain; IgG, immunoglobulin gamma; LC, light chain; PFD, prefolding dissociation; TCEP, tris(2-carboxyethyl) phosphine hydrochloride; TFA, trifluoroacetic acid. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.09.026

complete characterization of these products. Sequence level identification of modifications in bio-therapeutics is often carried out by using bottom-up approaches such as peptide mapping followed by LC/MS analysis [5–7]. Peptide mapping is an information-rich technique but suffers from the complexity of sample preparation and data analysis. In addition, the peptide mapping protocol itself can sometimes induce putative modifications. Recent advances in top-down fragmentation approaches have allowed sequence level identification of proteins and has provided an attractive alternative to conventional bottom-up techniques [8–14]. Top-down analysis has found applications in biomarker discovery [15–17] and is also being increasingly used for the identification of posttranslational modifications in proteins [8,11]. The application of top-down sequencing in bio-therapeutic characterization can be challenging because of the complexity of such data, low throughput, and low sensitivity. The sensitivity during top-down fragmentation can be improved with the prefolding dissociation (PFD) method recently developed by McLafferty and co-workers [10]. The dissociation in this method was carried out in the post skimmer region without selection of a specific precursor ion. PFD and other in-source dissociation methods do not allow the selection of a specific molecular ion for dissociation. Hence, the major challenge in using these methods in biopharmaceutical applications is the requirement to separate proteins and covalent modifications in proteins prior to analysis [18].

43

Top-down fragmentation of IgG molecules / D. Ren et al. / Anal. Biochem. 384 (2009) 42–48

In this paper, we report a method for the top-down N-terminal fragmentation of immunoglobulin gamma (IgG) subunits by combining reversed-phase separation on the diphenyl column followed by CAD with an electrospray ionization time-of-flight mass spectrometer. N-terminal sequencing is often used as an identity test for therapeutic IgG molecules. N-terminal sequencing also allows the identification of signal peptides left over during processing and detects modifications in the solvent-exposed N-terminal amino acids The methods described in this paper allow rapid top-down N-terminal sequence determination of up to seven terminal residues in IgG subunits and is adaptable with high-throughput applications.

Inlet probe

Ion guide 1

Ion guide 2

Cone

1 torr

Materials and methods Materials Trifluoroacetic acid (TFA) and guanidine hydrochloride (GdnHCl) were obtained from Pierce (Rockford, IL). Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) was obtained from Sigma (St. Louis, MO). HPLC grade water and acetonitrile (ACN) were obtained from VWR international (West Chester, PA). Pepsin and trypsin were obtained from Roche (Indianapolis, IN). The IgG lots were produced and purified using processes proprietary to Amgen and kept frozen at 80 °C until used. Reversed-phase chromatography The separation of IgG fragments was carried out on an HP1100 series CapLC system equipped with a Varian Diphenyl Pursuit, (2.1  150 mm column). Typically, 20 lg of protein was injected onto the column. The column was held at 95% solvent A (0.1% TFA in water) and 5% solvent B (90% acetonitrile, 10% water, and 0.1% TFA in water) for 5 min followed by a 7-min gradient from 5% B to 35% B. Antibody elution was achieved with a liner gradient from 35% B to 46% B in 40 min. The column temperature and flow rate were maintained at 80 °C and 200 lL/min throughout the run. Reduction of the IgG molecule Reduction was achieved by incubating 0.5 mL of IgG at a concentration of 2 mg/mL in denaturing buffer (7.5 M GdnHCl, 120 mM sodium acetate, pH 5.0) containing 5 mM TCEP, at 37 °C for 30 min. Mass spectrometry Mass spectrometric analysis was carried out on a Waters LCT Premier equipped with an ESI source. The analysis was carried out in positive W mode in which the ions are reflected through a reflectron to obtain an instrument resolution of approximately 10,000. The capillary and cone voltages were set at 2500 and 80 V, respectively. The desolvation and source temperatures were set at 350 and 80 °C, respectively. All the other voltages were optimized to provide maximal signal intensity in each of the modes. All raw data were processed using Waters Masslynx MaxEnt 1 software to obtain the deconvoluted mass.

Results and discussion Several mass spectrometer parameters were tested for their effectiveness in the fragmentation of antibody light (LC) and heavy chains (HC) separated with on-line reversed-phase chromatography on the diphenyl column. In-source fragmentation of proteins by elevating the cone voltage in Q-TOF instruments [19] or the

Region of CAD fragmentation Fig. 1. Schematic of in-source fragmentation in the LCT premier. The schematic of the LCT premier source showing the inlet probe, cone, ion guide 1 and ion guide 2.

nozzle skimmer dissociation (NSD) in FTMS and Orbitrap instruments [14,20,21] has been reported. In the LCT instrument used for this study, the cone voltage and the source temperature did not have a significant effect on the fragmentation of either the HC or the LC. The relatively high pressure in the cone region shown in Fig. 1 limits the effectiveness of the cone voltage in collisionally activated dissociation (CAD). The ion tunnels including Ion guide 1 and ion guide 2 are composed of stacks of rings [22] that can transfer gas-phase ions from the ion source to the TOF mass analyzer by the radiofrequency-only voltages applied on these rings. The CAD technique described in this paper occurs in the region between the ion guide 1 and the aperture separating the guides; see Fig. 1. In the ion guide 1 region, the protein ions gain significant internal energy as they accelerate. Collision of these ions with neutral molecules of the residual atmospheric gas leads to their dissociation. The effect of ion guide 1 voltage on the fragmentation of a 25-kDa IgG light chain separated with on-line diphenyl column reversed-phase chromatography is shown in Fig. 2. Multiply charged ions of the light chain ranging from +5 to +30 charge states can be seen at all the ion guide 1 voltages. Sodium and TFA adducts were observed at the lower voltage of 20 V especially on the lower charge states. Increasing the ion guide 1 voltage to 50 and 70 V led to the dissociation of the sodium adducts and improved the quality of the m/z spectrum. Increasing the ion guide voltage further caused fragmentation of the LC to generate a prominent sequential series of b-ions from the N-terminus of the molecule. This b-ion series was most predominant at an ion guide voltage of 100 V and allowed the N-terminal sequencing of the LC. Furthermore, at this voltage, there were still a sufficient number of multiply charged ions of the intact LC to allow the determination of the relative molecular mass of the intact LC. Increasing the ion guide voltage to 125 and 180 V caused further fragmentation, but reduced the intensity of the terminal b-ion series and also the multiply charged ions of the LC. Hence, an ion guide voltage of 100 V was selected for the CAD in further experiments as it would allow the determination of the relative molecular mass of the intact protein and at the same time yield N-terminal sequence information. Analysis of IgG heavy chains is more complex because of its larger size (50 kDa) and due to the presence of glycosylation. The CAD fragmentation of HC separated with on-line reversed-phase chromatography using a diphenyl column is shown in Fig. 3. It can be seen from Fig. 3, that similar to LC, HC fragmentation also predominantly produced the terminal b-ion series. A series of ions from b3 to b9 was detected. Along with the terminal b-ions, the

44

Top-down fragmentation of IgG molecules / D. Ren et al. / Anal. Biochem. 384 (2009) 42–48

Ion guide 1 voltage (V) 100

[M+7H]7+

995.97 1086.46 8+

[M+8H] [M+9H]9+

%

919.46

3428.89

20

[M+6H]6+

0

100 %

3412.93 3412.42 3413.54

2986.44

956.211039.27 1138.16

50

0

100

1195.05 1327.68 1593.22 1837.98

%

1086.49

3413.01

70

0

1195.07

%

100

1327.73 1493.54

b3 b4 b5 b2

3412.80 3411.98 3413.65

3981.52

100

3412.65 3411.95 3413.51

3981.43

125

3412.89 3412.13 3413.74

3981.66

180

0

%

100

b3 b4

342.17 455.29

b2

b5

1405.75 1493.60 1593.09

870.46

0

%

100 0

m/z 500

1000

1500

2000

2500

3000

3500

4000

4500

m/z Fig. 2. Influence of ion guide 1 voltages on the fragmentation of an IgG light chain (LC). Spectra for the IgG LC acquired under variable ion guide voltages of 20–180 V are shown. The predominant b-ions generated with an ion guide voltage of 100 and 125 V are labeled. Also labeled in the topmost spectrum are [M+H] the charge states for some of the most intense LC ions.

b ions Da 130 229 357 470 569 698 785 842 899

EVQLVESGGG G0 50290

100

b3

785

b5

357.16 470.26 569.35

G1 50453 %

100

b4

50069

50228 50533

0

b7 %

b6

49600

698.42 b9 785.47 902.52

[b45 + 4H]

49800

50000

50200

50400

50600

50800

51000

mass

4+

1165.66

1625.89

0

m/z 200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

m/z Fig. 3. Fragmentation of an IgG heavy chain (HC). Top-down fragmentation spectrum of an IgG HC using an ion guide voltage of 100 V is shown. The various b-ions from the HC are labeled. The inlay shows the deconvoluted spectrum for the HC obtained from the multiply charged ions. G0 and G1 refer to glycosylation variations.

quadruply charged b45 ion of HC was also observed. The b45 ion is a result of fragmentation between a leucine (L) and a glutamic acid (E) residue. The mechanism of fragmentation is not fully understood. Additionally, the multiply charged ions of HC observed

between m/z 1000 to m/z 2000 could be used to obtain its intact mass. The deconvoluted spectrum of the HC is shown in the inlay of Fig. 3. The multiple peaks observed in the deconvoluted spectrum were due to glycosylation heterogeneity, which has been

45

Top-down fragmentation of IgG molecules / D. Ren et al. / Anal. Biochem. 384 (2009) 42–48

described in detail previously [23]. This ability to obtain the molecular mass of the intact protein and fragmentation data provides a powerful tool for the characterization of proteins. The major requirement of CAD and other in-source fragmentation techniques is that the protein under investigation must be

highly pure. The purity of proteins can be further affected by common chemical modifications in protein side chains. A newly developed on-line diphenyl column separation was used with ESI to separate the HC, LC, and chemical modifications in these chains [24,25]. This separation allows selection of specific protein 35.72

LC

LC

A

HC

35.72

100

53.58

%

52.79

38.36 55.36

34.79

0

40.00

50.00

60.00

Time

%

30.00

LC2 38.36

LC3 34.79

LC 1

LC4 30.42 12

Time

30.00

31.00

32.00

33.00

34.00

35.00

36.00

37.00

38.00

39.00

40.00

41.00

Retention Time (min) 100 %

B

y7

b3

342.14 475.30 870.39

1099.59

3412.68 3412.30 3413.03 3454.11 3411.86

3981.33

LC4

3412.63 3412.06 3419.91 3446.11

3989.72

LC3

3412.01 3409.71 3418.84

3987.55

3410.22 3409.71 3410.60 3409.27 3411.07

3978.45

3412.65 3413.31 3413.74

3981.43

3500

4000

0

y7

b3

342.17

%

100

870.40 870.35

1099.61

100

y7

%

0

870.48

LC 1

0

100 %

y7 870.47

1404.60 1492.44

LC2

0

%

100

y7

b3 342.18 455.29

870.46

3411.95

1405.75 1493.60 1593.09

LC

0

m/z 500

1000

1500

2000

2500

m/z

3000

4500

Fig. 4. On-line LC/MS analysis of IgG subunits. (A) Detailed view for the separation of LC showing peaks LC1, LC2, LC3, LC4, and LC. The entire reversed-phase chromatogram of LC and HC from a reduced IgG molecule is shown in the inlay. (B) Fragmentation spectra of the LC peaks (from panel A) showing fragment ions as well as multiply charged envelopes. (C) Deconvoluted spectra of the LC variant peaks from (panel A) generated by processing the multiply charged envelope shown in (B) using MaxEnt 1 algorithm.

46

Top-down fragmentation of IgG molecules / D. Ren et al. / Anal. Biochem. 384 (2009) 42–48

LC4

23838

100 %

C

0

23838

100 %

LC3

0

23837

100

LC1

%

23819.62 23857.10

0

23818

100 %

LC2

0

23837

%

100

LC

0

mass 22000

22500

23000

23500

24000

24500

25000

25500

26000

Deconvoluted mass Fig. 4 (continued)

isoforms for subsequent top-down analysis. The reversed-phase separation of LC and HC fragments from an IgG molecule is shown in the Fig. 4A inlay. Several satellite peaks labeled as LC1-4 were observed in the chromatogram of the LC (Fig. 4A). These peaks were caused by chemical modifications of specific amino acid residues in the LC. The top-down m/z spectra generated for each of the chromatographic peaks are shown in Fig. 4B. Fragmentation information was obtained for most of the chromatographic peaks. Each mass spectrum also contains multiply charged envelopes. These multiply charged envelopes were deconvoluted to obtain the mass of the LC-related peaks. The deconvoluted spectra for the LC and satellite chromatographic peaks are show in Fig. 4C. The masses of all the satellite peaks except for peak LC2 were within a 1 or 2 Da error margin from the calculated mass of the main LC peak. These peaks can be contributed to chemical modifications such as isomerization of aspartic acid or deamidation of asparagine. The diphenyl reversedphase chromatographic analysis appears to resolve these chemical modifications from the unmodified main chain LC peak. The presence of such modifications was confirmed by bottom-up analysis of this IgG molecule (data not shown). In a previous study, we have shown that the diphenyl column is able to resolve site-specific modifications in IgG subunits [24,25]. Hence, the chromatographic peaks LC1, LC3, and LC4 could be a result of site-specific deamidation or isomerizations. In Fig. 4C, only the LC2 peak showed a 19 Da loss in mass from the main LC peak. Cyclization of the terminal glutamic acid into pyroglutamic acid leads to a mass shift of 18 Da, which has been previously reported in this molecule [26] and could lead to the generation of the LC2 peak. The top-down spectra of peaks LC1, LC3, and LC4

showed profiles similar to the spectrum of LC, which indicates that the observed heterogeneity was not N-terminal related. A detailed labeling of the zoomed-in regions of the mass spectra of LC and LC2 from Fig. 4B is shown in Fig. 5. N-terminal b-ions from b2 to b5 and C-terminal ions y7 to y9 were the predominant ions in the spectrum. The sequential b-ions were used for confirming the unmodified N-terminal sequence in the LC peak. The spectrum for the LC2 peak showed the unmodified y7 to y9 ions, confirming that the first nine C-terminal residues were unmodified. However, all the detectable b-ions showed a loss of 18 Da. The smallest b-ion in the LC2 peak was b3-18, which comprises the sequence EIV. The only known modification on that sequence that can lead to a 18 Da shift would be cyclization of the terminal glutamate (E) to pyroglutamic acid (pE). These data indicate that the chromatographic peak LC2 was caused by the cyclization of the terminal E to pE. Conclusions The field of top-down protein sequencing is rapidly growing and finding new applications in the fields of proteomics and protein characterization. Top-down studies usually utilize highresolution tandem mass spectrometers such as FT-ICR or Orbitraps and are usually carried out with off-line infusion of purified proteins. The CAD method described here relies on the chromatographic separation of site-specific modifications in the individual IgG chains, followed by on-line top-down fragmentation on a simple, nontandem, orthogonal ESI-TOF instrument. This method produces a prominent terminal b-ion series and at the same time allows for computation of the relative molecular mass of proteins.

Top-down fragmentation of IgG molecules / D. Ren et al. / Anal. Biochem. 384 (2009) 42–48 b ions Da

47

130 243 342 455 556 684

EIVLTQS

y7 y7

b3 b3

342.18

100

%

b4 b4

b2 b2

LC

870.44

455.29

b5 b5 556.36

243.10

b6

b6

y9 y9 y8 y8 1099.65

998.58

771.49

0 LC2

y7 y7 870.45

%

100

b3 -18 b3-18

324.16 0

200

b5-18 b5 -18 b4-18 b4 -18 538.34

b6-18 b6 -18 753.49

437.27 400

y9 y9

y8 y8 1099.66 998.58

600

800

1000

m/z

Fig. 5. Identification of cyclization of the N-terminal glutamic acid residue. Fragmentation spectra of the LC and LC2 chromatographic peaks from Fig. 4A. Various b and y-ions are labeled.

This b-ion series can be easily processed to obtain the N-terminal sequencing. The method as described here cannot be used to determine sequence and modifications in the hypervariable CDR regions and can only be used for the sequencing of about 7–10 terminal residues. These terminal sequences, however, define the beginning and the end of the polypeptide chain and, hence, can be used as an identity test for pharmaceutical products. These sequences are usually solvent exposed, and therefore, more susceptible to chemical modifications. In IgG molecules, modifications such as glycation [27], pyroglutamate conversion, amidation, and lysine variance [1] have been reported on the N and C termini. The method described here could be used as a high-throughput method for the rapid sequencing of IgG chains and for the detection of chemical modification in the terminal residues. Acknowledgments The authors thank Danielle Pace for proofreading and Dr. James Thomas for critical review of this manuscript.

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15] [16]

References [1] H.S. Gadgil, P.V. Bondarenko, G.D. Pipes, T.M. Dillon, D. Banks, J. Abel, G.R. Kleemann, M.J. Treuheit, Identification of cysteinylation of a free cysteine in the Fab region of a recombinant monoclonal IgG1 antibody using Lys-C limited proteolysis coupled with LC/MS analysis, Anal. Biochem. 355 (2006) 165–174. [2] H.S. Gadgil, G.D. Pipes, T.M. Dillon, M.J. Treuheit, P.V. Bondarenko, Improving mass accuracy of high performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of intact antibodies, J. Am. Soc. Mass Spectrom. 17 (2006) 867–872. [3] R.J. Harris, K.L. Wagner, M.W. Spellman, Structural characterization of a recombinant CD4-IgG hybrid molecule, Eur. J. Biochem. 194 (1990) 611–620. [4] W. Zhang, L.A. Marzilli, J.C. Rouse, M.J. Czupryn, Complete disulfide bond assignment of a recombinant immunoglobulin G4 monoclonal antibody, Anal. Biochem. 311 (2002) 1–9. [5] J. Bongers, J.J. Cummings, M.B. Ebert, M.M. Federici, L. Gledhill, D. Gulati, G.M. Hilliard, B.H. Jones, K.R. Lee, J. Mozdzanowski, M. Naimoli, S. Burman, Validation of a peptide mapping method for a therapeutic monoclonal antibody: what could we possibly learn about a method we have run 100 times?, J Pharm. Biomed. Anal. 21 (2000) 1099–1128. [6] K.A. Johnson, K. Paisley-Flango, B.S. Tangarone, T.J. Porter, J.C. Rouse, Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain 1, Anal. Biochem. 360 (2007) 75–83. [7] J.D. Ramos, N. Cheong, B.W. Lee, K.Y. Chua, Peptide mapping of immunoglobulin E and immunoglobulin G immunodominant epitopes of an

[17]

[18] [19]

[20]

[21] [22] [23]

[24]

allergenic Blomia tropicalis paramyosin, Blo t 11, Clin. Exp. Allergy 33 (2003) 511–517. Y. Ge, B.G. Lawhorn, M. ElNaggar, S.K. Sze, T.P. Begley, F.W. McLafferty, Detection of four oxidation sites in viral prolyl-4-hydroxylase by top-down mass spectrometry, Protein Sci. 12 (2003) 2320–2326. V. Zabrouskov, X. Han, E. Welker, H. Zhai, C. Lin, K.J. van Wijk, H.A. Scheraga, F.W. McLafferty, Stepwise deamidation of ribonuclease A at five sites determined by top down mass spectrometry, Biochemistry 45 (2006) 987– 992. X. Han, M. Jin, K. Breuker, F.W. McLafferty, Extending top-down mass spectrometry to proteins with masses greater than 200 kilodaltons, Science 314 (2006) 109–112. M.T. Boyne, J.J. Pesavento, C.A. Mizzen, N.L. Kelleher, Precise characterization of human histones in the H2A gene family by top down mass spectrometry, J. Proteome Res. 5 (2006) 248–253. Y. Du, B.A. Parks, S. Sohn, K.E. Kwast, N.L. Kelleher, Top-down approaches for measuring expression ratios of intact yeast proteins using Fourier transform mass spectrometry, Anal. Chem. 78 (2006) 686–694. C.E. Thomas, N.L. Kelleher, C.A. Mizzen, Mass spectrometric characterization of human histone H3: a bird’s eye view, J. Proteome Res. 5 (2006) 240– 247. J.A. Loo, C.G. Edmonds, R.D. Smith, Tandem mass spectrometry of very large molecules. 2. Dissociation of multiply charged proline-containing proteins from electrospray ionization 1, Anal. Chem. 65 (1993) 425–438. K. Cottingham, A new top-down proteomics workflow 9, J. Proteome Res. 6 (2007) 20. R.L. Graham, C.E. Pollock, N.G. Ternan, G. McMullan, Top-down proteomic analysis of the soluble sub-proteome of the obligate thermophile, Geobacillus thermoleovorans T80: insights into its cellular processes, J. Proteome Res. 5 (2006) 822–828. G. Scherperel, G.E. Reid, Emerging methods in proteomics: top-down protein characterization by multistage tandem mass spectrometry 2, Analyst 132 (2007) 500–506. B.T. Chait, Chemistry. Mass spectrometry: bottom-up or top-down?, Science 314 (2006) 65–66 J.M. Ginter, F. Zhou, M.V. Johnston, Generating protein sequence tags by combining cone and conventional collision induced dissociation in a quadrupole time-of-flight mass spectrometer, J. Am. Soc. Mass Spectrom. 15 (2004) 1478–1486. J.A. Loo, H.R. Udseth, R.D. Smith, Peptide and protein analysis by electrospray ionization-mass spectrometry and capillary electrophoresis-mass spectrometry, Anal. Biochem. 179 (1989) 404–412. Z. Zhang, B. Shah, Characterization of variable regions of monoclonal antibodies by top-down mass spectrometry, Anal. Chem. 79 (2007) 5723–5729. S. Guan, A.G. Marshall, Stacked-ring electrostatic ionguide, J. Am. Soc. Mass Spectrom. 7 (1996) 101. S. Krapp, Y. Mimura, R. Jefferis, R. Huber, P. Sondermann, Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity, J. Mol. Biol. 325 (2003) 979–989. D. Ren, G.D. Pipes, D.M. Hambly, P.V. Bondarenko, M.J. Treuheit, D.N. Brems, H.S. Gadgil, Reversed-phase liquid chromatography of immunoglobulin G molecules and their fragments with the diphenyl column 2, J. Chromatogr. A 1175 (2007) 63–68.

48

Top-down fragmentation of IgG molecules / D. Ren et al. / Anal. Biochem. 384 (2009) 42–48

[25] D. Ren, G. Pipes, G. Xiao, G.R. Kleemann, P.V. Bondarenko, M.J. Treuheit, H.S. Gadgil, Reversed-phase liquid chromatography-mass spectrometry of sitespecific chemical modifications in intact immunoglobulin molecules and their fragments, J. Chromatogr. A 1179 (2008) 198–204. [26] D. Chelius, K. Jing, A. Lueras, D.S. Rehder, T.M. Dillon, A. Vizel, R.S. Rajan, T. Li, M.J. Treuheit, P.V. Bondarenko, Formation of pyroglutamic acid from N-

terminal glutamic acid in immunoglobulin gamma antibodies, Anal. Chem. 78 (2006) 2370–2376. [27] H.S. Gadgil, P.V. Bondarenko, G. Pipes, D. Rehder, A. McAuley, N. Perico, T. Dillon, M. Ricci, M. Treuheit, The LC/MS analysis of glycation of IgG molecules in sucrose containing formulations, J. Pharm. Sci. 96 (2007) 2607–2621.