Use of Lysozyme as a Standard for Evaluating the Effectiveness of a Proteomics Process

Automated sequencing of unknowns in bottom-up proteomics makes the data produced susceptible to process control errors, which can be propagated into mistakes in analyte identification. Inclusion of an unintrusive internal standard, such as lysozyme, allows monitoring all phases of the proteomics process including sample preparation, enzymatic digestion, HPLC, mass spectrometry, and database searching. By using this internal standard, digestion issues including rearrangements, semi-tryptic fragments, and modifications were monitored. In addition, control of the HPLC process including column performance was achieved. The use of the lysozyme standard allowed easy optimization of mass spectral conditions including data dependent and collision induced dissociation settings. The use of this internal standard in a study of differential protein expression in rat serum samples is presented. Keywords: data-dependent • LC−MS • internal standard • proteomics • HPLC • mass spectrometry