pH/Acetonitrile-Gradient Reversed-Phase Fractionation of Enriched Hyper-Citrullinated Library in Combination with LC-MS/MS Analysis for Confident Identification of Citrullinated Peptides

Abstract

Citrullination, the Ca²⁺-driven enzymatic conversion of arginine residues to citrulline, is a posttranslational modification, implicated in several physiological and pathological processes. Several methods to detect citrullinated proteins have been developed, including color development reagent, fluorescence, phenylglyoxal, and antibody-based methods. These methods yet suffer from limitations in sensitivity, specificity, or citrullinated site determination. Mass spectrometry (MS)-based proteomic analysis has emerged as a promising method to resolve these problems. However, due to low abundance of citrullinated proteins and similar MS features to deamidation of asparagine and glutamine, confident identification of citrullinated proteome is challenging. Here, we present a systematic approach to identify a compendium of steps to enhance the number of detected citrullinated residue and implement diagnostic MS feature that allow the confidence of MS-based identifications. Our method is based on the concept of generation of hyper-citrullinated library with high-pH reversed-phase peptide fractionation that allows to enrich in low abundance citrullinated peptides and amplify the effect of charge loss upon citrullination. Application of our approach to complex global citrullino-proteome datasets demonstrates the confident assessment of citrullinated peptides, thereby enhancing the size and functional interpretation of citrullinated proteomes.

Keywords: Citrullination; Deamidation; Hyper-citrullinated peptide library; Mass spectrometry; Retention time; pH reversed-phase peptide fractionation.

Fig. 1

Workflow to generate hyper-citrullinated peptide library. Proteins in each individual sample were pooled (comprised of equal portions of individual samples) and divided into two samples. Following FASP steps just after IAA treatment, samples were conditioned with deamidation buffer to allow citrullination by incubation with PADs. Following citrullination and washing steps, samples were digested, and fractionated using two types of high-pH fractionation methods. Every prepared sample was analyzed on a Q Exactive mass spectrometer

Fig. 2

Hyper-citrullinated library from THP-1 macrophages: number of citrullinated peptides and sites with and without PAD enrichment (PAD+ versus PAD−)

Fig. 3

Workflow for identification and validation of citrullinated sites: (in anti-clockwise direction, starting from the top right panel) The workflow begins with (a) sample preparation followed by (b) data acquisition and conversion from raw format to open-source format. (c) The fragment ion spectra are searched against a protein sequence database for the identification of PSMs and scored. (d) High-scoring PSMs are then converted to spectral library, which is then used by (e) CitFinder for the identification and validation of citrullinated peptides. Further, after generation of consensus library from the spectral library, analysis using available SWATH tools can be done

Fig. 4

Schematic depiction of categorization of modified–unmodified peptide pairs using Skyline: Chromatographic peaks are characterized on the basis of factors such as Isotope Dot Product, Retention Time Drift, Total Area, Total Area/FWHM, and Average Mass Error PPM to be defined as (a) “Good,” (b) “Okay,” and (c) “Bad”