Identification of prolyl hydroxylation modifications in mammalian cell proteins - PubMed (original) (raw)

Identification of prolyl hydroxylation modifications in mammalian cell proteins

Patrick R Arsenault et al. Proteomics. 2015 Apr.

Abstract

Prolyl hydroxylation is a PTM that plays an important role in the formation of collagen fibrils and in the oxygen-dependent regulation of hypoxia inducible factor-α (HIF-α). While this modification has been well characterized in the context of these proteins, it remains unclear to what extent it occurs in the remaining mammalian proteome. We explored this question using MS to analyze cellular extracts subjected to various fractionation strategies. In one strategy, we employed the von Hippel Lindau tumor suppressor protein, which recognizes prolyl hydroxylated HIF-α, as a scaffold for generating hydroxyproline capture reagents. We report novel sites of prolyl hydroxylation within five proteins: FK506-binding protein 10, myosin heavy chain 10, hexokinase 2, pyruvate kinase, and C-1 Tetrahydrofolate synthase. Furthermore, we show that identification of prolyl hydroxylation presents a significant technical challenge owing to widespread isobaric methionine oxidation, and that manual inspection of spectra of modified peptides in this context is critical for validation.

Keywords: Cell biology; MS; PTM; Prolyl hydroxylase domain protein; Prolyl hydroxylation.

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Conflict of interest statement

Conflicts of Interest: The authors declare no conflict of interest.

Figures

Figure 1

Figure 1

Outline of sample preparation and enrichment strategies. (A) Direct analysis of unfractionated tryptic digests. (B) Following tryptic digests but prior to LC injection, peptides are fractionated by isoelectric focusing (IEF). (C) Fractionation of intact proteins by gel filtration chromatography, followed by tryptic digest prior to LC injection (GF). (D) VHL capture probe-based enrichment of hydroxylated peptides.

Figure 2

Figure 2

(A) Outline of results of computational analyses. Diagram shows results of Tide-based hydroxyproline search. Largest circle indicates the set of unique peptides that are called with at least one oxidation event (i.e. Proline, Methionine, etc.) as either the most likely, or when applicable, the second most likely candidate for a given spectra (i.e. first and second ranked peptide by XCorr score). Successive filtering steps are indicated. (B) Patterns of enrichment for amino acids immediately adjacent to primary site of assigned prolyl hydroxylation. Values presented are derived from the set of 185 unique peptides identified by Tide analyses as being likely prolyl hydroxylated.

Figure 3

Figure 3

Representative fragmentation spectra (MS2) for Collagen type I, α1 chain. The b and y ions are indicated only when ions appeared above a 2-fold signal to noise ratio. Ions appearing in +2 charge states are indicated with “++”; all others are singly charged species.

Figure 4

Figure 4

(A) Comparison of overall peptide and putative hydroxyproline peptide identification rates between Tide- and MaxQuant-based analyses platforms at the 1% FDR level. All values refer to numbers of unique peptides. “Putative Hydroxyproline Peptides” refers to the total set of peptides identified as being prolyl hydroxylated by the given analysis package prior to subtraction of likely confounding methionine oxidation and/or manual inspection of fragment spectra. Comparison of (B) average number of oxidations per unique modified peptide and (C) average length of both modified and unmodified peptides, as identified by MaxQuant and Tide analyses respectively. Bars indicate +/- 1 SD, and * indicates p < 0.05 by Student's t-Test.

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