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 ϳ3000 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. Shotgun proteomics is a widely used and evolving method for determining the protein composition of a biological mixture (1-3). It most often involves the digestion of denatured proteins by trypsin, followed by the identification of product peptides and the use of this information to infer protein identities and possibly targeted post-translational modifications (PTMs)1 . However, because digestion is a highly complex chemical process, a large proportion of identifiable products are not specifically targeted for analysis and therefore invisible to the analysis. These include unexpected and unwanted peptides that interfere with the analysis. Others may contain modifications of biological origin, which, unless specifically targeted, can be lost among the forest of artifacts (4 -6). This paper describes methods for building a tandem mass spectral library capable of characterizing all identifiable peptides in a tryptic digest of a selected protein. Spectral libraries are known to provide an effective way of reusing this information to quickly, reliably, and sensitively determine peptide identities (7-11). These identifications can serve several purposes, including 1) ensuring that all previously identified peptides are identified regardless of search engine settings, 2) tagging artifact peptides that might otherwise lead to false positive identifications, 3) ensuring the identification of known and identifiable biological post-translational modifications without explicitly looking for them, and 4) providing a list of artifact peptides for assessing the quality of the sample preparation process.HSA, human serum albumin, was selected as the target protein for library development partly because of its ubiquity, making up Ͼ50% of the total protein in blood (12-13) and therefore found in many biological samples, and partly because of the considerable background information available

Development of Diagnostic Fragment Ion Library for Glycated Peptides of Human Serum Albumin: Targeted Quantification in Prediabetic, Diabetic, and Microalbuminuria Plasma by Parallel Reaction Monitoring, SWATH, and MSE

“…However, it is important to quantify CML-and CELmodified albumin, as these modifications constitute the predominant AGEs. Several mass-spectrometry-based targeted quantification approaches, including multiple reaction monitoring, parallel reaction monitoring, and SWATH, have become powerful tools and rely heavily on fragment ion library (39,40). In this context, we constructed a fragment ion library for the synthetically AML-, CML-, and CEL-modified peptides of albumin by using high-resolution accurate mass spectrometer followed by rigorous inspection and validation of MS/MS spectra.…”

Modified cysteine S-phosphopeptide standards for mass spectrometry-based proteomics

“…However, for longer and poorly fragmented peptides it was not always possible to locate multiple modifications unambiguously. More detailed analysis of the model Digest 1 would certainly be possible, by additional HPLC-runs and alternative MS-fragmentation strategies (Dong et al 2014).…”

“…It is important to mention that proteomic samples may contain traces of thiol groups that emerged from various precursors. In general, the presence of free thiols is mainly associated with incomplete alkylation of thiol groups from reducible sulfhydryl modifications (Dong et al 2014;Muller and Winter 2017;Suttapitugsakul et al 2017). Moreover, even traces of S-acylated peptides can generate Cys-peptides as a result of acyl transfer events (Ji et al 2013(Ji et al , 2016.…”

Spectral library searching in proteomics

“…Dong et al. presented an algorithm for creating spectral libraries for specific proteins . As an example, they created a spectral library for Serum Albumin.…”

“…Although this dataset is intended for the creation of SRM assays, it can also be used to create targeted spectral libraries. Dong et al presented an algorithm for creating spectral libraries for specific proteins [40]. As an example, they created a spectral library for Serum Albumin.…”