Strategies in relative and absolute quantitative mass spectrometry based proteomics.

Quantitative mass spectrometry approaches are used for absolute and relative quantification in global proteome studies. To date, relative and absolute quantification techniques are available that differ in quantification accuracy, proteome coverage, complexity and robustness. This review focuses on most common relative or absolute quantification strategies exemplified by three experimental studies. A label-free relative quantification approach was performed for the investigation of the membrane proteome of sensory cilia to the depth of olfactory receptors in Mus musculus. A SILAC-based relative quantification approach was successfully applied for the identification of core components and transient interactors of the peroxisomal importomer in Saccharomyces cerevisiae. Furthermore, AQUA using stable isotopes was exemplified to unraveling the prenylome influenced by novel prenyltransferase inhibitors. Characteristic enrichment and fragmentation strategies for a robust quantification of the prenylome are also summarized.

Low-bias phosphopeptide enrichment from scarce samples using plastic antibodies.

Phosphospecific enrichment techniques and mass spectrometry (MS) are essential tools for comprehending the cellular phosphoproteome. Here, we report a fast and simple approach for low sequence-bias phosphoserine (pS) peptide capture and enrichment that is compatible with low biological or clinical sample input. The approach exploits molecularly imprinted polymers (MIPs, "plastic antibodies") featuring tight neutral binding sites for pS or pY that are capable of cross-reacting with phosphopeptides of protein proteolytic digests. The versatility of the resulting method was demonstrated with small samples of whole-cell lysate from human embryonic kidney (HEK) 293T cells, human neuroblastoma SH-SY5Y cells, mouse brain or human cerebrospinal fluid (CSF). Following pre-fractionation of trypsinized proteins by strong cation exchange (SCX) chromatography, pS-MIP enrichment led to the identification of 924 phosphopeptides in the HEK 293T whole-cell lysate, exceeding the number identified by TiO2-based enrichment (230). Moreover, the phosphopeptides were extracted with low sequence bias and showed no evidence for the characteristic preference of TiO2 for acidic amino acids (aspartic and glutamic acid). Applying the method to human CSF led to the discovery of 47 phosphopeptides belonging to 24 proteins and revealed three previously unknown phosphorylation sites.