Direct measurement of light and heavy antibody chains using ion mobility and middle-down mass spectrometry

Abstract

The analysis of monoclonal antibodies (mAbs) by a middle-down mass spectrometry (MS) approach is a growing field that attracts the attention of many researchers and biopharmaceutical companies. Usually, liquid fractionation techniques are used to separate mAbs polypeptides chains before MS analysis. Gas-phase fractionation techniques such as high-field asymmetric waveform ion mobility spectrometry (FAIMS) can replace liquid-based separations and reduce both analysis time and cost. Here, we present a rapid FAIMS tandem MS method capable of characterizing the polypeptide sequence of mAbs light and heavy chains in an unprecedented, easy, and fast fashion. This new method uses commercially available instruments and takes ~24 min, which is 40-60% faster than regular liquid chromatography-MS/MS analysis, to acquire fragmentation data using different dissociation methods.

Deeper Protein Identification by Using FAIMS in Top-down Proteomics

“…Analysis of TDS with FAIMS and employing a CV of -30V resulted in a waveform that favored the stable trajectory of only ubiquitin as shown on the second panel of Figure 1. As the CV was increased by 10V in subsequent LC injections, the remaining protein standards were observed with larger protein species generally preferring more positive CVs, consistent with previous observations 21 (Figure 1). The exception among the standards was trypsinogen, which contains disulfide bonds.…”

“…Its use in TDP analysis has been limited, with most investigators studying intact protein standards. Investigators employing ion-mobility-time-offlight mass spectrometry systems have shown that IMS influences the arrival time distribution of different peptides, denatured protein charge states and multimeric native protein structures [17][18][19][20] Our previous study employed static electrospray ionization (ESI) coupled with a commercial implementation of FAIMS to separate and analyze a simple mixture of heavy and light chains from a monoclonal antibody 21 . Without gas-phase fractionation, the light chain dominates the precursor ion signal in MS experiments, resulting in low signal-to-noise and poor detection of the heavy chain.…”

“…Without gas-phase fractionation, the light chain dominates the precursor ion signal in MS experiments, resulting in low signal-to-noise and poor detection of the heavy chain. Utilization of FAIMS prior to MS resolved the two antibody chains by favoring the FAIMS stability of the heavy or light chain of the antibody while suppressing the other, all as a function of the applied compensation voltage 21 . Additionally, combining liquid extraction surface analysis (LESA) with FAIMS provided a clear improvement in signal-to-noise of intact precursor ion signal compared to the same LESA samples analyzed without FAIMS [22][23][24][25][26] .…”

Inline electrochemical reduction of NISTmAb for middle-up subunit liquid chromatography-mass spectrometry analysis

“…Localization of PTMs to an individual subunit is also beneficial for the assessment of critical quality attributes. [8][9][10] Subunit production is often carried out by non-biological chemical reaction and/or enzyme digestion. 10,11 Consistent subunit production is essential in middle-up and middledown mass spectrometry workflows requiring effective reduction steps.…”

Enhancing top-down proteomics of brain tissue with FAIMS

“…34 To prevent collision of the ions with the electrode, a deviation in the ion's path is introduced through the application of a DC compensation voltage (CV), 35 allowing selective transmission of that ion. 34 The application of FAIMS to intact protein analysis has largely been restricted to the separation of conformers of individual proteins or small combinations of proteins, [36][37][38][39][40][41] however, FAIMS has recently been applied to intact and native protein analyses using liquid extraction surface analysis (LESA). [42][43][44][45][46] Here we apply FAIMS-TDP analysis to a whole-tissue sample from the medial frontal cortex (MFC) of an Alzheimer's patient.…”