Improved in-solution tryptic digestion of proteins in terms of speed and peptide coverage was achieved with the aid of a novel acid-labile anionic surfactant (ALS). Unlike SDS, ALS solubilizes proteins without inhibiting trypsin or other common endopeptidases activity. Trypsin activity was evaluated in the presence of various denaturants; little or no decrease in proteolytic activity was observed in 0.1-1% ALS solutions (w/v). Sample preparation prior to mass spectrometry and liquid chromatography analysis consists of sample acidification. ALS degrades rapidly at low-pH conditions, which eliminates surfactant-caused interference with analysis. Described methodology combines the advantages of protein solubilization, rapid digestion, high peptide coverages, and easy sample preparation for mass spectrometry and liquid chromatography analyses.

In recent years, the use and number of biotherapeutics has increased significantly. For these largely protein-based therapies, the quantitation of aggregates is of particular concern given their potential effect on efficacy and immunogenicity. This need has renewed interest in size-exclusion chromatography (SEC). In the following review we will outline the history and background of SEC for the analysis of proteins. We will also discuss the instrumentation for these analyses, including the use of different types of detectors. Method development for protein analysis by SEC will also be outlined, including the effect of mobile phase and column parameters (column length, pore size). We will also review some of the applications of this mode of separation that are of particular importance to protein biopharmaceutical development and highlight some considerations in their implementation.

Silylation chemistry on porous silicon provides for ultrahigh sensitivity and analyte specificity with desorption/ionization on silicon mass spectrometry (DIOS-MS) analysis. Here, we report that the silylation of oxidized porous silicon offers a DIOS platform that is resistant to air oxidation and acid/base hydrolysis. Furthermore, surface modification with appropriate hydrophobic silanes allows analytes to absorb to the surface via hydrophobic interactions for direct analyte extraction from complex matrixes containing salts and other nonvolatile interferences present in the sample matrix. This enables rapid cleanup by simply spotting the sample onto the modified DIOS target and removing the liquid phase containing the interferences. This approach is demonstrated in the analysis of protein digests and metabolites in biofluids, as well as for the characterizing of inhibitors from their enzyme complex. An unprecedented detection limit of 480 molecules (800 ymol) for des-Arg(9)-bradykinin is reported on a pentafluorophenyl-functionalized DIOS chip.

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