Quantitation of Human Peptides and Proteins Via MS: Review of Analytically Validated Assays

Abstract

Since the development of monoclonal antibodies in the 1970s, antibody-based assays have been used for the quantitation of proteins and peptides and, today, they are the most widely used technology in routine laboratory medicine and bioanalysis. However, in the last couple of decades, liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) techniques have been adopted in the quantitation of small molecules, and more recently have made significant contributions in the quantitation of proteins and peptides. In this article, we will review clinical MS-based assays for endogenous peptides, proteins, and therapeutic antibodies, for which validated methods exist. We will also cover the measurement of protein turnover and the unique solutions that MS can offer in this field.

An Overall Comparison of Small Molecules and Large Biologics in ADME Testing

“…Differently from SMs, the most commonly used bioanalytical methods for LMs are ELISA based assays by either direct or bridging ELISA measuring the concentration of the therapeutic over time in plasma or serum for in vivo studies case by case. In comprehensive reviews and white papers, several bioanalytical methods, considerations and strategies as well as challenges have extensively been discussed for LMs [42,46,[60][61][62][63][64][65][66]. This section will provide an overview on recent advancements in bioanalysis of mAbs, ADC, in particular with ELISA and LC-MS/MS technologies and related new approaches dealing with matrix interference.…”

“…Moreover, a versatile immunoaffinity LC-MS/MS method was developed to quantify total receptor activator of nuclear factor-kappaB ligand (RANKL) in the presence of denosumab, a humanized monoclonal antibody (mAb) specific to RANKL with an LLOQ of RANKL down to 3.13 ng/mL for mouse plasma PK, which was not able to determine the total RANKL because the interference of denosumab decreased the recovery of RANKL with commercial ELISA kit [81]. Additionally, 2D-LC (LC×LC)-MS/MS methodology with improved MS detection limit of approximately two orders of magnitude over direct LC-MS/MS was recommended [82], as well as other LC-MS formats and approaches such as capillary LC-MS gaining MS sensitivity were reviewed [66], offering new opportunities in the analysis of monoclonal antibodies.…”

The clinical utility of mass spectrometry based protein assays

“…The use of chromatography is especially important to eliminate or reduce ion suppression and matrix effects [22]. The analysis of peptides and proteins is conducted almost exclusively with liquid chromatography [23][24][25][26][27][28][29] rather than gas chromatography as peptides are inherently polar (and able to accept protons directly) and cannot enter the gas phase. However, the use of matrix-assisted-laser-desorption (MALDI) [30] and MS imaging [31] for clinical analysis have also been reported, but neither of these techniques will be described at length in this review.…”

Absolute and Multiplex Quantification of Antibodies in Serum Using Psaq™ Standards and LC-MS/MS

“…Although relative reproducibility can be obtained using these standards, the accuracy of quantification is affected by loss of proteins during sample prefractionation stages or proteolysis steps. Recently, alternative strategies using a more ideal full-length labeled mAb standard were described [22,25]. These labeled mAb standards, described as generic or universal, can be introduced early in the analytical workflow, overcoming process variability and thus making accurate absolute quantification possible.…”