Rapid and Quantitative Enrichment of Peptides from Plasma for Mass Spectrometric Analysis.

Mass spectrometric analysis of peptides enables the assignment of their exact mass and confirmation of all or a significant portion of the peptide's amino acid sequence. LC-MS/MS analysis has proven invaluable in peptidomics research and can identify new biomarkers and assign their circulatory concentrations to aid research into disease processes. However, due to the high background plasma protein content, which masks the presence of the naturally low abundance circulatory peptidome, extraction of peptides from plasma prior to mass spectrometric analysis is therefore crucial. Organic solvents efficiently precipitate these high molecular weight plasma proteins while leaving small molecular weight peptides in solution, providing a rapid and effective technique for separating peptides from the contaminating plasma proteins. A secondary cleanup step involving solid phase extraction is required to remove lipids and highly hydrophobic contaminants before LC-MS/MS analysis. The method described within this chapter is effective at enriching circulatory plasma peptides prior to LC-MS/MS analysis and has been used in multiple peptidomic studies to improve peptide detection and quantification. Peptides studied using this methodology include insulin, C-peptide, glucagon, PYY, GIP, and a number of other challenging gut peptide hormones. Quantitative analyses of peptides using the described method showed good correlation with existing immunoassays.

Associations between Plasma Branched Chain Amino Acids and Health Biomarkers in Response to Resistance Exercise Training Across Age.

Leucine, isoleucine and valine (i.e., the branched chain amino acids, BCAA) play a key role in the support and regulation of tissue protein regulation and also as energy substrates. However, positive relationships exist between elevated levels of BCAA and insulin resistance (IR). Thus, we sought to investigate the links between fasting plasma BCAA following a progressive resistance exercise training (RET) programme, an intervention known to improve metabolic health. Fasting plasma BCAA were quantified in adults (young: 18-28 y, n = 8; middle-aged: 45-55 y, n = 9; older: 65-75 y, n = 15; BMI: 23-28 kg/m2, both males and females (~50:50), in a cross-sectional, intervention study. Participants underwent 20-weeks whole-body RET. Measurements of body composition, muscle strength (1-RM) and metabolic health biomarkers (e.g., HOMA-IR) were made pre- and post-RET. BCAA concentrations were determined by gas-chromatography mass spectrometry (GC-MS). No associations were observed across age with BCAA; however, RET elicited (p < 0.05) increases in plasma BCAA (all age-groups), while HOMA-IR scores reduced (p < 0.05) following RET. After RET, positive correlations in lean body mass (p = 0.007) and strength gains (p = 0.001) with fasting BCAA levels were observed. Elevated BCAA are not a robust marker of ageing nor IR in those with a healthy BMI; rather, despite decreasing IR, RET was associated with increased BCAA.