Overcoming biofluid protein complexity during targeted mass spectrometry detection and quantification of protein biomarkers by MRM cubed (MRM3).

Targeted mass spectrometry in the so-called multiple reaction monitoring mode (MRM) is certainly a promising way for the precise, accurate, and multiplexed measurement of proteins and their genetic or posttranslationally modified isoforms. MRM carried out on a low-resolution triple quadrupole instrument faces a lack of specificity when addressing the quantification of weakly concentrated proteins. In this case, extensive sample fractionation or immunoenrichment alleviates signal contamination by interferences, but in turn decreases assay performance and throughput. Recently, MRM(3) was introduced as an alternative to MRM to improve the limit of quantification of weakly concentrated protein biomarkers. In the present work, we compare MRM and MRM(3) modes for the detection of biomarkers in plasma and urine. Calibration curves drawn with MRM and MRM(3) showed a similar range of linearity (R(2) > 0.99 for both methods) with protein concentrations above 1 µg/mL in plasma and a few nanogram per milliliter in urine. In contrast, optimized MRM(3) methods improve the limits of quantification by a factor of 2 to 4 depending on the targeted peptide. This gain arises from the additional MS(3) fragmentation step, which significantly removes or decreases interfering signals within the targeted transition channels.

What sample preparation should be chosen for targeted MS monoclonal antibody quantification in human serum?

AIM: Monoclonal antibody-based treatment of cancer has been established as one of the most successful therapeutic strategies. MATERIALS & METHODS: In this work, we developed a workflow based on an automated protein-A capture and LC-MS/MS analysis to quantify bevacizumab on patient serum during treatment. This analytical approach was fully validated and compared with a commercially available Monoclonal antibody-based treatment preparation (nanosurface and molecular-orientation limited kit). RESULTS: The analytical comparison of the two preparative workflows based on protein-A capture gave similar results with a better lower limit of quantification for the nanosurface and molecular-orientation limited kit (0.26986 vs 1.9565 µg/ml). CONCLUSION: LC-MS/MS has clear advantages compared with ELISA when considering method development time, multiplexing capacities and absolute quantification with internal standardization.

Absolute quantification of podocin, a potential biomarker of glomerular injury in human urine, by liquid chromatography-multiple reaction monitoring cubed mass spectrometry.

Glomeruli play a major role in the kidney function since they are involved in primary urine formation. It is then crucial to dispose of methods to monitor glomerular injury, especially in drug development. In this context, quantification of podocin could be of great interest since it is a protein exclusively present in highly specialized glomerulus cells called podocytes. Immunoassays are the most commonly used approach for protein assays. However, they rely on the availability of specific antibodies. When such antibodies are not available, liquid chromatography tandem mass spectrometry (LC-MS/MS), in selected reaction monitoring (SRM) or in multiple reaction monitoring cubed (MRM(3)) mode, has been demonstrated as a powerful alternative technique, and can be applied to multiple protein quantification. This paper describes the development of a quantification method of human podocin in urine by LC-MS/MS in MRM(3) mode. Inter assay precision and accuracy ranged from 7 to 20% and from 105 to 112% respectively and the lower limit of quantification (LLOQ) was 0.39ng/mL from only 1mL of urine which is compatible for endogenous level of podocin determination.

Optimization of liquid chromatography-multiple reaction monitoring cubed mass spectrometry assay for protein quantification: application to aquaporin-2 water channel in human urine.

Aquaporin-2 (AQP2) is a water channel protein located in the kidney collecting ducts that has been studied as a potential biomarker of a wide variety of water handling disorders and that could also be used to monitor lesions in the collecting ducts. Enzyme-linked immunosorbent assay (ELISA), the most commonly used approach for protein assay in biofluids, has a limited potential for biomarker verification due to the restricted possibility to perform multiplex assays, the cost and complexity of assay development for new candidates. Liquid chromatography tandem mass spectrometry (LC-MS/MS), in multiple reaction monitoring (MRM) mode, has been demonstrated as a powerful alternative technique, and applied to multiple protein quantification. An even more specific method, termed MRM cubed (MRM(3)), has recently been developed. This paper focuses on the development of an AQP2 assay in urine by LC-MS/MS, based on the MRM(3) strategy, and the influence of key MRM(3) parameters that enable to increase the method sensitivity by a factor of 10. Linearity is observed within the concentration range 0.5-50ng/mL, intra and inter assay precision ranged from 9 to 35% at the lower limit of quantification (LLOQ), and accuracy from 94 to 114%. This assay could therefore be used in the near future to evaluate human urinary AQP2 as a potential biomarker of kidney collecting duct injury.

The current status of clinical proteomics and the use of MRM and MRM(3) for biomarker validation.

The transfer of biomarkers from the discovery field to clinical use is still, despite progress, on a road filled with pitfalls. Since the emergence of proteomics, thousands of putative biomarkers have been published, often with overlapping diagnostic capacities. The strengthening of the robustness of discovery technologies, particularly in mass spectrometry, has been followed by intense discussions on establishing well-defined evaluation procedures for the identified targets to ultimately allow the clinical validation and then the clinical use of some of these biomarkers. Some of the obstacles to the evaluation process have been the lack of the availability of quick and easy-to-develop, easy-to-use, robust, specific and sensitive alternative quantitative methods when immunoaffinity-based tests are unavailable. Multiple reaction monitoring (MRM; also called selected reaction monitoring) is currently proving its capabilities as a complementary or alternative technique to ELISA for large biomarker panel evaluation. Here, we present how MRM(3) can overcome the lack of specificity and sensitivity often encountered by MRM when tracking minor proteins diluted by complex biological matrices.