Intact protein bioanalysis by liquid chromatography-High-resolution mass spectrometry

摘要

Liquid chromatography coupled online to mass spectrometry (LCMS) is arguably the most widely used bioanalytical technique due to its sensitivity and selectivity.It is a very versatile analytical approach that can be tuned to address a wide range of molecules with applications from environmental analysis to drug analysis and doping control.LCMS is increasingly used for the analysis of proteins, typically after a proteolytic digestion step which converts the macromolecular analytes into lower-molecular weight peptides, which can be readily quantified.LC-MS analysis of intact proteins, however, is still in its infancy, being mainly restricted to the detailed characterization of biopharmaceuticals in dose formulations [1–10].The analysis of intact proteins in complex biological samples, referred to here as protein bioanalysis, has remained the realm of ligand binding assays (LBAs) and notably of enzyme-linked immunosorbent assays (ELISAs) due to their exquisite sensitivity and specificity.A disadvantage of ELISAs is that specificity can often not be assessed due to a lack of alternative analytical techniques and the fact that the binding site is not known (or not provided by the manufacturer).Despite considerable advances in mass spectrometry and liquid chromatography, protein bioanalysis by LC-MS remains challenging due to the fact that a given (set of) protein(s) needs to be detected in a sensitive and selective manner in a matrix of (hundreds of) thousands of other proteins.The task is further complicated by the fact that proteins are not very amenable to LC separation and that they generate complex mass spectra upon electrospray ionization (ESI), the most widely used ionization method for protein analysis by LC-MS, due to multiple charge states (so-called charge-state envelopes).Quantitative protein bioanalysis, notably in the area of biopharmaceuticals, would, however, benefit from analysis at the intact protein rather than at the peptide level, since protein spec