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Distinguishing between Leucine and Isoleucine by Integrated LC-MS Analysis using an Orbitrap Fusion Mass Spectrometer Yongsheng Xiao, Malgorzata Monika Vecchi, and Dingyi Wen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03409 • Publication Date (Web): 05 Oct 2016 Downloaded from http://pubs.acs.org on October 9, 2016

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Analytical Chemistry

Distinguishing between Leucine and Isoleucine by Integrated LCMS Analysis using an Orbitrap Fusion Mass Spectrometer

Yongsheng Xiao, Malgorzata M. Vecchi, Dingyi Wen*

Analytical Biochemistry, Department of Cell and Protein Sciences, Biogen, 225 Binney Street, Cambridge, Massachusetts 02142

*To whom correspondence should be addressed: [email protected]. Tel.: (617) 679-2362; Fax: (617)-679-3208.

1 ACS Paragon Plus Environment


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Abstract Despite the great success of mass spectrometry (MS) for de novo protein sequencing, Leu and Ile have been generally considered to be indistinguishable by MS because their molecular masses are exactly the same. Positioning of incorrect Leu/Ile residues in variable domains, especially in CDRs (complementarity determining regions) of an antibody, may result in substantial loss of antigen binding affinity and specificity of the antibody. Here, we describe an integrated LC-MS based strategy, encompassing a combination of HCD (high-energy collisional dissociation) multistage mass spectrometric analysis (HCD-MSn) and ETD (electron transfer dissociation)-HCD MS3 analysis using an Orbitrap Fusion mass spectrometer, to reliably identify Leu and Ile residues in proteins and peptides.

The merits and limitations of this Leu/Ile

discrimination approach are evaluated. Using the new approach, along with proposed decisionmaking guidelines we unambiguously identified every Leu/Ile residue in peptides containing up to five Leu/Ile residues and molecular masses up to 3000 Da.

In addition, we have

demonstrated, for the first time, that every Leu/Ile residue in the variable regions of a monoclonal antibody that could not be assigned by antibody germline sequence alignment could be correctly determined using this approach. Our results suggest that, by incorporating this approach into existing de novo antibody sequencing protocols, 100% of antibody amino acid sequences, including identity of Leu and Ile residues, can be accurately obtained solely by means of mass spectrometry.

In principle, this integrated, online LC-MS approach for Leu/Ile

assignment can be applied to de novo sequencing of any protein or peptide.


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Introduction Development of protein therapeutics, especially monoclonal antibodies (mAbs), has gained significant momentum in recently years.1,2 Antibody-based therapy has become one of the most successful and indispensable strategies for treating patients with a variety of severe diseases, including inflammatory3 and autoimmune4 diseases, as well as cancer5.

The amino acid

sequence of a mAb, especially the amino acids in the complementarity determining regions (CDRs), will influence the affinity and specificity of the mAb for its antigen and may also affect the efficacy and toxicity of a mAb drug6. Although the amino acid sequence of an antibody is mainly obtained by DNA sequencing of source cell line7, sometimes de novo sequencing of an antibody is required, as in the case where original cell line or cDNA is not available.8,9 Traditionally, Edman degradation coupled with proteolytic digestion has been utilized for protein and peptide sequencing; however, this method has low-throughput and low sensitivity, and is time-consuming. During the past 20 years, developments in instrumentation and bioinformatic tools have made mass spectrometry (MS) the method of choice for comprehensive protein characterization6,10 and global protein quantification.11,12 In terms of de novo antibody sequencing, multi-enzyme digestion followed by high resolution mass spectrometry analysis has been demonstrated to be a reliable approach to determine antibody sequence with very high accuracy.8,9 Free13 and commercially available14 software have also been developed to facilitate the automatic assembly of overlapping peptide segments into intact light and heavy chain sequences of antibodies. However, Leu (leucine) and Ile (isoleucine) residues are generally considered to be indistinguishable by MS because their molecular masses are exactly the same; thus Edman degradation often has to be used to distinguish between these two isomeric residues.



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Considerable efforts have been made over the years to solve this problem using mass spectrometric analysis. Some years ago, high-energy collision activated dissociation (HE CAD) methods that relied on the observation of unique w- or d-ions from Leu/Ile residues were reported for Leu/Ile discrimination.15,16 Despite some success, the HE CAD method has been proved not to be attractive because the very complicated MS2 spectra and the low intensity of targeted w- or d-ions, made Leu/Ile assignment not entirely reliable. Moreover, HE CAD can be implemented only on specialized mass spectrometers such as magnetic sector or TOF-TOF instruments; magnetic sector instruments are no longer being manufactured and samples cannot be analyzed online with TOF-TOF instruments. In 2007, a low energy collision-induced dissociation multistage mass analysis method, CID MSn, was developed17 for Leu/Ile discrimination based on unique fragmentation pattern of immonium ions18. The method exhibited good potential, but it only worked for peptides with molecular masses less than 1600 Da17, since CID MSn experiments were carried out in an ion trap type mass spectrometer, in which low-mass end fragment ions were lost during the fragmentation process of large precursor ions.19 Furthermore, the CID MSn method often fails when the Leu/Ile is located adjacent to a C-terminal lysine because of the inefficient generation of immonium ions under CID condition.17 In 2003, Kjeldsen et al. demonstrated that diagnostic w-ions can also be produced by hot electron capture dissociation (HECD) from secondary side chain loss of z-ions containing Nterminal Leu/Ile.20 The method was successfully utilized to distinguish Leu from Ile in a novel hemoglobin variant in 2009.21 Recently, Lebedev et al. utilized a similar concept and developed an ETD-HCD MS3 method for Leu/Ile discrimination using an Orbitrap Fusion mass spectrometer equipped with electron transfer dissociation (ETD) and high-energy collisional


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dissociation (HCD) functions.22 Despite being very effective in many ways, the ETD-HCD MS3 method still has some limitations. For example, it requires the production of critical z-ions with Leu/Ile at the N-terminus. To overcome these limitations, an alternative strategy is required for full discrimination of Leu/Ile residues in proteins/peptides. Here, we report an integrated strategy that combines a HCD multistage mass analysis (HCD MSn) method with an ETD-HCD MS3 method for unambiguous discrimination of Leu/Ile residues. We demonstrated, for the first time, that reliable Leu/Ile assignment for complex protein digest mixtures can be achieved by online LC-MS analysis on an Orbitrap Fusion mass spectrometer using this approach. The method is very sensitive, reliable, and quick. Using this strategy, every Leu/Ile identity in peptides containing up to five Leu/Ile residues and molecular mass of 3000 Da can be easily determined online.

In addition, using this online LC-MS

platform, coupled with multi-enzymatic digestion, we rapidly determined every Leu and Ile residue that could not be assigned by the antibody germline sequence alignment in the variable regions of antibodies.

This online LC-MS Leu/Ile discrimination approach can be readily

implemented into current de novo sequencing protocols so that complete amino acid sequences of antibodies or other proteins can be conclusively determined by means of mass spectrometry alone.

Materials and Experimental Procedures Materials The peptide mixture kit used in this study was purchased from Sigma-Aldrich (MSRT1). Monoclonal mouse antibody standard was purchased from Waters Corp. (PN 186006552). In-solution multi-enzyme digestion of antibody



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Reduction and alkylation. About 50 µg of the antibody was reduced with 27 mM DTT in 6 M guanidine hydrochloride, 100 mM Tris, pH 7.5, 5 mM EDTA, at 37 oC for 40 min in a total volume of 113 µL. The reduced antibody was then alkylated by adding 7 µL of 1 M of iodoacetamide (IAM) solution to a final concentration of 64 mM and incubating at room temperature in the dark for 45 min. The reaction was stopped by adding 1 µL of 1 M DTT. The reduced and alkylated protein was distributed into vials with 3.7 µg per vial. Protein in each vial was recovered by precipitation with a 40x volume of chilled ethanol for 1 h at -20 oC and then centrifuged at 14000g at 4 °C for 10 min. The supernatant was discarded and the precipitate was washed once with cooled ethanol.

The protein pellet was re-dissolved in 80 µL of 50%

acetonitrile and dried in a Speed-Vac. Digestion of antibody with multiple endo-proteases. The protein precipitates in three vials, each containing ~3.7 µg of the reduced and alkylated antibody, were reconstituted in 2 M urea, 0.2 M Tris-HCl, pH 7.4, 2 mM CaCl2, 5 mM MgCl2 solution; the protein in one vial was digested with trypsin (Promega) (5% w/w), the second with endo-LysC protease (Promega) (5% w/w) (both at room temperature for 8 h), and the third vial with chymotrypsin (Roche) (3% w/w) at room temperature for 2 h. The final digestion volume in each case was 25 µL. LC-MS Analysis All LC-MS/MS analysis was performed on a nanoLC-MS system comprised of a nanoACQUITY UPLC (Waters Corp.) and an Orbitrap Fusion mass spectrometer equipped with a nano-electrospray ionization source (Thermo Fisher Scientific). About 500 fmol of the peptide standard or 500 fmol of a digest of the antibody was loaded automatically onto a nanoACQUITY UPLC Symmetry C18 trap column (180 µm × 20 mm) by a Waters nanoAcquity UPLC system at a flow rate of 3 µL/min. The trapping column was connected to a 25-cm fused silica


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analytical column (PicoTip Emitter, New Objective, 75 µm i.d.) with 3 µm C18 beads (ReproSilPur 120 C18-AQ, Dr. Maisch). Peptides were then separated with a 60-min linear gradient of 10-43% acetonitrile containing 0.1% formic acid at a flow rate of 300 nL/min. The spray voltage was set at 1.7 kV to obtain a stable nanospray. To ensure that the precursors of interest will be triggered for fragmentation in MSn experiments and to maximize MS detection sensitivity, the Orbitrap Fusion was operated in the targeted data analysis mode to determine Leu/Ile identity. To set up the targeted mode analysis or the “tMSn” analysis in the Orbitrap Fusion, the “tMSn” experimental tab was dragged and dropped into the method panel, and then the m/z value and charge state of a precursor were input for each MSn stage. In the ETD-HCD MS3 experiment, all tandem mass spectra were acquired in the Orbitrap mass analyzer with resolution of 60,000. The ETD reaction time ranging from 60-110 ms with CID supplemental activation was set according to peptide size, and the ETD automatic gain control target was set to 4 × 105.

HCD

fragmentation at normalized collision energy (NCE) 12 was then applied to generate diagnostic w-ions from selected z-ions. For HCD multistage mass analysis (HCD MSn), optimized collision energy (NCE 28-40) was applied to multistage fragmentation (HCD MS2, HCD MS3 or HCD MS4 ) of Leu/Ile-containing fragment ions to produce 86-Da Leu/Ile immonium ions, which were then subjected to further HCD fragmentation at NCE 18 to generate diagnostic 69-Da ions. MSn spectra were acquired in an ion trap analyzer to maximize the sensitivity. The automatic gain control target was set to 4 × 105 with a maximum injection time of 300 ms, and 2-5 microscans were accumulated, depending on the order of the multistage fragmentations. Edman Degradation Peptides of interest in a digest of the antibody were collected manually from a UPLC (ACQUITY Waters Corp.) using an ACQUITY HSS T3 C18 column (1.8-μm particle size, 2.1



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mm x 150 mm; Cat# 186003540, Waters Corp.) for separation with a 75-min water/acetonitrile gradient (0-70% acetonitrile) containing 0.03% TFA at a flow rate of 0.07 mL/min at 30 ºC. A PVDF membrane was pre-treated with BioBrene solution, and then about 50 pmol of each peptide was loaded on the membrane and dried. Edman degradation was carried out on an Applied Biosystems Procise 494 High Throughput (HT) sequencer, which was run in the pulsed liquid PVDF mode. The resulting phenylthiohydantoin (PTH) amino acids were separated using an ABI 140C Microgradient System with a PTH C18 Column (2.1 mm x 220 mm) and analyzed on-line using a Perkin Elmer Series 200 UV/Vis detector. The data were analyzed using the ABI Sequence Pro version 2.1 data analysis software.

Results and Discussion LC-MS analysis workflow. Several mass spectrometry-based approaches to distinguishing Leu/Ile residues in peptides have been reported.

Among them, tandem mass spectrometry analysis with multistage

fragmentation (CID MSn)17 and ETD-HCD MS3 strategies22 show significant potential due to their high specificity and simplicity. However, both methods have limitations. For example, the CID MSn method does not work for a peptide with m/z >1600 Da or for Leu/Ile that is located next to C-terminal lysine.17 On the other hand, the ETD-HCD MS3 method requires critical zions with Leu/Ile at the N-terminus, and it may not work for z-ions with extremely large or small m/z values. To overcome these limitations, we developed an integrated and systematic online LC-MS analysis strategy for Leu/Ile determination based on these methods but with critical modifications and optimizations (Figure 1). Because the limitation of the CID MS n method for large peptides was due to the use of an ion trap for CID fragmentation, we reasoned that the


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application of HCD fragmentation in conjunction with a similar MSn protocol might greatly improve the performance of MSn analysis.

High-energy collisional dissociation (HCD)

fragmentation in Orbitrap mass spectrometers was introduced a few years ago and provides full mass range tandem mass analysis capability without the low-mass cutoff23. In addition, the beam type fragmentation feature of HCD induces more efficient ion fragmentation than does low energy CID. Fortunately, HCD multistage mass analysis (HCD MSn) has been implemented as a standard feature of the latest Orbitrap Fusion and Orbitrap Fusion Lumos mass spectrometers.24 The excellent ion transfer efficiency and detector sensitivity of Orbitrap Fusion instrument 25 also help make LC-MS analysis for Leu/Ile assignments routine and reliable. On the other hand, despite some limitations, the ETD-HCD MS3 method is very powerful for Leu/Ile discrimination, especially for peptides containing more than one Leu/Ile residue. As we show here, both the HCD MSn and EtD-HCD MS3 methods can be implemented simultaneously on an Orbitrap Fusion mass spectrometer equipped with a nanoLC system, with no need for special hardware modification. As a proof of concept, we first tested the reliability and specificity of our proposed online LC-MS protocol (Figure 1) by identifying every Leu or Ile residue in the component peptides of a commercially available peptide standard mixture having a wide-range of amino acid compositions and a varied number of Leu/Ile residues (Table 1). HCD MS3 analysis for peptides containing a single Leu/Ile residue A previously reported17 CID MSn approach relies on fragmentation of the 86-Da immonium ion of Leu or Ile to generate distinctive fragmentation patterns: fragmentation of the 86-Da immonium ion from Ile will give rise to a high abundance of diagnostic 69-Da ions, whereas the 86-Da immonium ion from Leu produces little (1000 Da and that alternative multiply charged z-ions with lower m/z values may be required to distinguish Leu and Ile. However, if multiply charged z-ions for large peptides are not detected, the ETD-HCD MS3 method will fail for Leu/Ile discrimination. For instance, the ETD-HCD MS3 method failed to confirm the identity of Ile7 in peptide TDELFQIEGLKEELAYL, because only the singly charged characteristic z12+ ion at m/z 1427.8 Da was observed. Second, since the success of ETD-HCD MS3 method relies heavily on the formation of critical z-ions with Leu/Ile at the Nterminus, the failure to generate highly abundant z-ions will inevitably preclude the use of the method. This limitation also applies to Leu/Ile residues located near the C-terminus of the peptide because diagnostic z-ions with m/z

Distinguishing between Leucine and Isoleucine by ... - ACS Publications

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