Mass and top-down analyses of 150-kDa monoclonal immunoglobulin gamma (IgG) antibodies were performed on an Orbitrap analyzer. Three different sample delivery methods were tested including (1) infusion of an off-line desalted IgG sample using nano-electrospray; (2) on-line desalting followed by a step elution with a high percentage of organic solvent; and (3) reversed-phase HPLC separation and on-line mass and top-down analyses of disulfide isoforms of an IgG2 antibody. The accuracy of mass measurements of intact antibody was within Ϯ2 Da (15 ppm). The glycoforms of intact IgG antibodies separated by 162 Da were baseline resolved. In-source fragmentation of the intact antibodies produced mainly 115 residue fragments including N-terminal variable domains of heavy and light chains. The sequence coverage (the number of cleavages) was greatly increased after reduction of disulfide bonds and HPLC/MS/MS analysis of light and heavy chains using collisioninduced dissociation in the ion trap of the LTQ-Orbitrap. This is an attractive alternative to peptide mapping for characterization and monitoring of post-translational modifications attributed to minimal sample preparation, high speed of the mass/top-down analysis, and relatively minor method-induced sample modifications. [1,2]. This has resulted in a strong need for a highthroughput methods for analysis of different antibody drug candidates. Mass spectrometry has become one of the most powerful techniques for the structural characterization of monoclonal antibodies (mAbs) [3]. Traditionally, structural characterization of mAbs has been performed by a "bottom-up" approach after first digesting them to peptides [4 -6]. Unfortunately, enzymatic digestion is a laborious, time-consuming process and it often introduces artificial modifications, such as cyclization of N-terminal glutamine and deamidation [5,7]. Alternatively, protein molecular mass analysis is relatively fast, does not require lengthy sample preparation, and induces fewer, if any, modifications [8] compared with the peptide mapping [5,7]. Analysis of intact monoclonal IgG antibodies and their large domains has been reported for matrix-assisted laser desorption/ ionization (MALDI) and electrospray ionization (ESI) sources and almost all mass analyzers including MALDI-time of flight (TOF) [9 -11], ESI quadrupole (Q) [4,[12][13][14], ion trap [15], orthogonal TOF [8,11,16,17], and the LTQ-Orbitrap during direct infusion [18]. Although a mass change in a population of mAbs can be used to monitor post-translational modifications [8], it cannot reveal the site of modification. For that purpose, fragmentation of intact proteins, also known as "topdown" mass spectrometry, has been developed during the past decade [19 -25]
The considerable progress in high throughput proteomics analysis via liquid chromatography-electrospray ionization-tandem mass spectrometry over the last decade has been fueled to a large degree by continuous improvements in instrumentation. High throughput identification experiments are based on peptide sequencing and are largely accomplished through the use of tandem mass spectrometry, with ion trap and trap-based instruments having become broadly adopted analytical platforms. To satisfy increasingly demanding requirements for depth of characterization and throughput, we present a newly developed dual-pressure linear ion trap mass spectrometer (LTQ Velos) that features increased sensitivity, afforded by a new source design, and demonstrates practical cycle times two times shorter than that of an LTQ XL, while improving or maintaining spectral quality for MS/MS fragmentation spectra. These improvements resulted in a substantial increase in the detection and identification of both proteins and unique peptides from the complex proteome of Caenorhabditis elegans, as compared to existing platforms. The greatly increased ion flux into the mass spectrometer in combination with improved isolation of low-abundance precursor ions resulted in increased detection of low-abundance peptides. These improvements cumulatively resulted in a substantially greater penetration into the baker’s yeast (Saccharomyces cerevisiae) proteome compared to LTQ XL. Alternatively, faster cycle times on the new instrument allowed for higher throughput for a given depth of proteome analysis, with more peptides and proteins identified in 60 min using an LTQ Velos than in 180 min using an LTQ XL. When mass analysis was carried out with resolution in excess of 25,000 FWHM, it became possible to isotopically resolve a small intact protein and its fragments, opening possibilities for top down experiments.
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