Characterizing citrullination by mass spectrometry-based proteomics

Abstract

Citrullination is an important post-translational modification (PTM) of arginine, known to play a role in autoimmune disorders, innate immunity response and maintenance of stem cell potency. However, citrullination remains poorly characterized and not as comprehensively understood compared to other PTMs, such as phosphorylation and ubiquitylation. High-resolution mass spectrometry (MS)-based proteomics offers a valuable approach for studying citrullination in an unbiased manner, allowing confident identification of citrullination modification sites and distinction from deamidation events on asparagine and glutamine. MS efforts have already provided valuable insights into peptidyl arginine deaminase targeting along with site-specific information of citrullination in for example synovial fluids derived from rheumatoid arthritis patients. Still, there is unrealized potential for the wider citrullination field by applying MS-based mass spectrometry approaches for proteome-wide investigations. Here we will outline contemporary methods and current challenges for studying citrullination by MS, and discuss how the development of neoteric citrullination-specific proteomics approaches still may improve our understanding of citrullination networks. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Keywords: citrullination; mass spectrometry; post-translational modifications; proteomics.

Figure 1.

Proteins are extracted from a biological sample such as cell culture or organs and subjected to proteolytic lysis to generate peptides. The peptides are separated by reversed-phase liquid chromatography coupled to the mass spectrometer (MS) and enter the MS as ionized peptides achieved by electrospray ionization. A full scan of the peptide mixture is recorded. Precursors ions are selected for fragmentation and fragment ions are recorded to generate the MS/MS scans. The full scan and MS/MS scans are analysed to obtain peptide identification and PTM localization, and bioinformatic analysis may be performed to gain additional biological insights.

Figure 2.

PTM enrichment allows for the analysis of only modified peptides.

Figure 3.

MS quantification strategies based on metabolic labelling (SILAC), chemical labelling (TMT) and label-free (LFQ). During SILAC samples are distinguished at the MS1 level according to the stable isotopic amino acid labelling. TMT labelled samples are distinguished at the MS1 level according to the stable isotopic amino acid labelling. TMT labelled samples are distinguished at the MS2 levels owing to the chemical tags resulting in different cleavage ions during fragmentation. During LFQ samples are run separately on the MS and are compared during the data processing level.

Figure 4.

The mass shift caused by citrullination is very similar to the naturally occurring shift caused of carbon-13 (¹³C) and nitrogen-15 substitution of the arginine residue.

Figure 5.

(a) Loss of isocyanic acid from citrulline peptide during fragmentation causing neutral loss of 43.006 Da. (b) Annotated MS/MS spectrum of citrulline-containing peptide and zoomed selection, demonstrating the mass shift of citrulline from b₂ to b₃ ion and matching y₁₅ and y₁₆, neutral loss of isocyanic acid from the citrulline b₃ to b_3* and similarly from y₁₆ to y_16*. Additionally, the immonium ion of citrulline at 130.09 m/z is also detected and highlighted. Blue, b-ions; red, y-ions; orange, z-ions; grey, unassigned. Spectra obtained from Rebak et al. [43].

Figure 6.

(a) Illustration of the citrulline effect which visualizes increased propensity of fragmentation following citrulline. (b) Baseline unmodified peptide. The y₁₃ ion contributes 0.5% to the total ion intensity. (c) Citrullinated peptide demonstrating the citrulline effect, exemplified by y₁₃ ion contributing 18% of the total ion intensity.