Data-independent acquisition (DIA) is becoming more prominent as a method for comprehensive proteomic analysis of clinical samples due to its ability to acquire essentially all fragment ion spectra in a single LC-ESI-MS/MS experiment. Since the direct correlation between a precursor and its fragment ions is lost when acquiring all ions in a defined m/z range, one data analysis strategy is using so-called peptide spectral libraries. These are usually generated by measuring similar biological samples in data-dependent (DDA) mode. The peptide spectral library content is a major limitation for the successful identification from DIA data. This is because a fragment ion spectrum from the sample can only be matched, and thus identified, when it is present in the peptide spectral library. In order to enhance peptide spectral library size, the sample for generating the peptide spectral library can be subjected to extended separation strategies prior to DDA. These strategies are of special relevance for biological samples containing a few very high-abundant proteins, such as CSF, as they enlarge the identification of low-abundant proteins. In instances of CSF separation, suitable methods include the 1D SDS-PAGE of proteins and high-pH reversed-phase peptide fractionation. Both methods are based on different protein/peptide characteristics, are complementary with one another, and are inexpensive and easy to establish. Ideally, DDA spectra from samples generated with both methods combine to achieve a comprehensive spectral library.
Keywords: DIA; High-pH reversed phase; In-gel digest; In-solution digest; Peptide fractionation; SDS-PAGE; Spectral library.