PhD: Study of protein biomarkers in biological fluids using immunological and mass spectrometry methods

Olgica Trenchevska
Institute of Chemistry, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University, Skopje, Macedonia
December, 2011


Proteins play important role in biological processes, since in their native form or specific isoform serve as potential biomarkers. The impetus for finding new biomarkers has been accelerated by the arrival of the “omics” technologies. However, equally important is the rediscovery of existing biomarkers with these new approaches as novel variants can be discovered that can improve their utility. Therefore, developing new and rapid methods for analysis of proteins and their isoforms is of great importance for determining their role in the onset of disease, progression and response to therapy.

The study presented here is a part of a larger effort to develop methods in order to understand protein diversity and specificity of a human proteome in health and disease. A need for such a research occurs due to the arguable fact that new techniques and methods for protein analyses have been developing, and only a few have actually been implemented in routine clinical practice.

Mass spectrometry, coupled to immunoaffinity separations, can provide an efficient mean for simultaneous detection and quantification of protein variants. In this work, we have used a novel mass spectrometry immunoassay (MSIA) method to analyze the intrinsic properties of several proteins. MSIA is a two-step assay for quantitative analysis of proteins and protein variants in biological samples. In the first step, immunoaffinity pipettes were used to isolate the protein of interest from the biological sample. Then, MALDI-TOF-MS was utilized to analyze and quantify the protein. Using this methodology, fully quantitative assays for beta-2-microglobulin, cystatin C, transthyretin and transferrin were developed and characterized in terms of their precision, linearity and recovery characteristics. The novel methods were compared to conventional ELISAs. Then, the assays were utilized to determine the individual concentration of protein variants across larger cohorts of samples, demonstrating the ability to fully quantify all individual forms of post-translationally modified proteins and point mutations. Additional proteolytic digestions using trypsin and endoproteinase Arg-C were performed for samples containing genetic heterogeneity in order to identify the point mutations. The digestion processes were further improved by introducing microwave assistance.

The novel mass spectrometric immunoassay method provided a fast, accurate and high-throughput quantitative analysis for all proteins when compared to conventional ELISAs. This method allowed detection and quantitation of protein isoforms, which can be used as part of specific protein biomarker discovery/rediscovery undertaking and can be a step forward in protein analysis in clinical practice.
These types of assays can find use in quantifying specific protein modifications, especially in the main effort - to aid in the process of unrevealing the protein complexity of neurological disorders and contribute to its better understanding.