Recent advances in absolute quantification of peptides and proteins using LC-MS

Current bioanalytical methods for the quantification of peptides and proteins used in biomedical research and pharmaceutical R&D are predominantly based on immunoassay methodologies, such as enzyme-linked immunosorbent assays (ELISA). Although these antibody-based techniques are exceptionally sensitive, they suffer from issues, such as cross-reactivity, long development time, and high variability. Recently, liquid chromatography-mass spectrometry (LC-MS)-based quantification techniques for large molecules, including the bottom-up and top-down approaches, are gaining popularity due to their shorter development time, higher specificity, and ability to detect degradation products and post-translational modifications (PTMs). The multiple reaction monitoring (MRM)-based bottom-up LC-MS/MS approach, which targets enzymatic digestion-produced signature peptides as surrogates in a quantification assay, is a sophisticated method with good sensitivity, selectivity, and multiplex capacity, although it may miss critical information such as PTMs, sequence variants and isoforms, and truncation and degradation products. The LC-high resolution mass spectrometry (LC-HRMS)-based top-down approach quantifies intact target molecules, thus avoiding the shortcomings of the bottom-up approach. The current LC-HRMS approach is most suitable for peptides and proteins smaller than 10–15 kDa, though that may change with the development of more advanced mass spectrometers with higher resolving power and broader mass ranges. The drawbacks and bottlenecks of LC-MS-based large molecule quantification assays are low sensitivity, complicated sample preparation, and relatively low throughput. Technology advancement may address these challenges and expand their suitable applications. In this article, the recent advances and trends in this emerging field are reviewed, along with the basic workflows, analytical approaches, and other common issues