Sample Preparation for LC‐MS Bioanalysis of Proteins

Sample preparation is an essential part of any LC-MS-based method for the quantitative determination of proteins in biological samples. Currently, most LC-MS methods employ an enzymatic digestion step to convert the protein analyte into a mixture of peptides and quantify one or more of these as a surrogate for the intact protein. Low-μg/mL to high-ng/mL concentrations can typically be measured if a digested sample is directly analyzed with LC-MS/MS. To improve selectivity and sensitivity, extraction of the protein before digestion and/or extraction of the surrogate peptide after digestion can be applied. Extraction approaches based on immunocapture provide the best selectivity and sensitivity, but require specific analyte-directed reagents such as antibodies and are not generic. Analyte-free extraction methods such as solid-phase extraction are a universally applicable but less selective alternative. By combining different, carefully optimized extraction techniques, low- to sub-pg/mL sensitivities can be reached. Digestion of the untreated biological matrix results in obtaining the total concentration of a protein analyte, while protein extraction before digestion might result in the free or total fraction or a concentration in between the two, depending on the extraction principle.