Tandem mass spectral libraries of peptides in digests of individual proteins: Human Serum Albumin (HSA)

Abstract

This work presents a method for creating a mass spectral library containing tandem spectra of identifiable peptide ions in the tryptic digestion of a single protein. Human serum albumin (HSA(1)) was selected for this purpose owing to its ubiquity, high level of characterization and availability of digest data. The underlying experimental data consisted of ∼3000 one-dimensional LC-ESI-MS/MS runs with ion-trap fragmentation. In order to generate a wide range of peptides, studies covered a broad set of instrument and digestion conditions using multiple sources of HSA and trypsin. Computer methods were developed to enable the reliable identification and reference spectrum extraction of all peptide ions identifiable by current sequence search methods. This process made use of both MS2 (tandem) spectra and MS1 (electrospray) data. Identified spectra were generated for 2918 different peptide ions, using a variety of manually-validated filters to ensure spectrum quality and identification reliability. The resulting library was composed of 10% conventional tryptic and 29% semitryptic peptide ions, along with 42% tryptic peptide ions with known or unknown modifications, which included both analytical artifacts and post-translational modifications (PTMs) present in the original HSA. The remaining 19% contained unexpected missed-cleavages or were under/over alkylated. The methods described can be extended to create equivalent spectral libraries for any target protein. Such libraries have a number of applications in addition to their known advantages of speed and sensitivity, including the ready re-identification of known PTMs, rejection of artifact spectra and a means of assessing sample and digestion quality.

Fig. 1.

Single-protein spectral library building pipeline. Flow diagram illustrating the six major stages of library building process used in the single-protein spectral library.

Fig. 2.

Distribution of nine peptide classes along the amino acid sequence of the protein. At each amino acid position is given the summed peptide ion identification frequency (PIIF) from all peptide classes containing that amino acid. Simple tryptic in blue (Class 1), Expected missed-cleavage in red (Class 2), Common modification in yellow (Class 3), In-source semitryptic in purple (Class 4), In-solution semitryptic in orange (Class 5), Artifact and PTM in light blue (Class 6), Unexpected missed-cleavage in green (Class 7), Under/over alkylation in green-blue (Class 8), and Unidentified modification in pink (Class 9).