Analysis of human C1q by combined bottom-up and top-down mass spectrometry: detailed mapping of post-translational modifications and insights into the C1r/C1s binding sites

Abstract

C1q is a subunit of the C1 complex, a key player in innate immunity that triggers activation of the classical complement pathway. Featuring a unique structural organization and comprising a collagen-like domain with a high level of post-translational modifications, C1q represents a challenging protein assembly for structural biology. We report for the first time a comprehensive proteomics study of C1q combining bottom-up and top-down analyses. C1q was submitted to proteolytic digestion by a combination of collagenase and trypsin for bottom-up analyses. In addition to classical LC-MS/MS analyses, which provided reliable identification of hydroxylated proline and lysine residues, sugar loss-triggered MS(3) scans were acquired on an LTQ-Orbitrap (Linear Quadrupole Ion Trap-Orbitrap) instrument to strengthen the localization of glucosyl-galactosyl disaccharide moieties on hydroxylysine residues. Top-down analyses performed on the same instrument allowed high accuracy and high resolution mass measurements of the intact full-length C1q polypeptide chains and the iterative fragmentation of the proteins in the MS(n) mode. This study illustrates the usefulness of combining the two complementary analytical approaches to obtain a detailed characterization of the post-translational modification pattern of the collagen-like domain of C1q and highlights the structural heterogeneity of individual molecules. Most importantly, three lysine residues of the collagen-like domain, namely Lys(59) (A chain), Lys(61) (B chain), and Lys(58) (C chain), were unambiguously shown to be completely unmodified. These lysine residues are located about halfway along the collagen-like fibers. They are thus fully available and in an appropriate position to interact with the C1r and C1s protease partners of C1q and are therefore likely to play an essential role in C1 assembly.

Fig. 1.

Schematic representation of structural organization of human C1q and of C1 assembly. A, model showing the CLR and globular heads of C1q and the association with the C1r/C1s tetramer forming the C1 complex. B, representation of the association between the A, B, and C chains leading to formation of the six ABC heterotrimeric triple helices of C1q CLR. The two following interruptions in the Gly-Y-Z triplet repeats are involved in the kink of the collagen-like region, i.e. the insertion of a threonine at position 39 in the A chain and the replacement of a glycine by an alanine at position 36 in the C chain (only the interruption in the A chain is shown).

Fig. 2.

Peptide identified by LC-MS/MS analyses of C1q successively digested with collagenase and trypsin using QqTOF and LTQ-Orbitrap instruments. Green lines, peptides identified in both laboratory-purified and commercial C1q; black and violet lines, peptides only identified in either laboratory-purified or commercial C1q, respectively; blue lines, peptides specifically identified from the “collagenase + semitrypsin” database search; dotted line (in C1q C), sequence GLPGPK#GEP* identified from the “collagenase + trypsin” search was finally rejected in favor of sequence GPK#GEPGIP* (collagenase + semitrypsin) because the latter better matched the experimental spectrum. Blue squares, hydroxylation; red squares, Glc-Gal modification; green squares, deamidation; empty square, pyrrolidone cyclization. When precise localization of one (two) hydroxylation(s) could not be established from MS/MS spectra, an extended striped blue/white rectangle overlapping the possibly modified residues (Pro or Lys) is shown.

Fig. 3.

Summary of post-translational modifications determined by bottom-up and top-down analyses of CLR and C1q samples. Black line, identifications from MS² data; gray line, identification from MS³ data; green line, identification from top-down data; no line, merged information on PTMs from bottom-up and top-down analyses. Fully blue squares, residue always detected as hydroxylated; blue/white squares, residue detected either as unmodified or hydroxylated, indicating partial modification; red squares, lysine always detected in a glycosylated form; red/blue squares, lysine detected either as glycosylated or hydroxylated. ? indicates that ambiguity remains as to whether Lys¹⁰ or Lys¹¹ of C1q A is glycosylated.

Fig. 4.

Sequences identified from MS³ scans acquired during LC-MS/MS analyses including sugar loss-triggered MS³ scans performed on both C1q and CLR samples. Dotted line, one disaccharide lost between consecutive MS² and MS³ scans; full line, two disaccharides lost. The color code is the same as in Fig. 2; additionally, orange lines, identifications from the CLR sample. Circled in red, lysines determined to be glycosylated from these MS²/MS³ analyses; circled in black, unmodified lysines.

Fig. 5.

Mass spectra of reduced CLR and C1q acquired by direct nano-ESI infusion of the proteins on LTQ-Orbitrap instrument. A, MS spectrum obtained on reduced CLR analyzed at a protein concentration of 5 pmol/μl. B, MS spectrum obtained on reduced C1q analyzed at a protein concentration of 5 pmol/μl.

Fig. 6.

Deconvoluted MS spectra obtained by top-down MS analysis of reduced CLR (A) and reduced C1q (B).

Fig. 7.

Deconvoluted and deisotoped MS/MS spectrum obtained from fragmentation of ionic species at about m/z 817.35 (14+) of CLR C1q A. # represents a Glc-Gal moiety, and * represents a hydroxylation. The 33 most intense ions were manually interpreted even though the deconvolution algorithm happened to be unsuccessful in determining the monoisotopic mass (errors of 1 mass unit). The most intense fragment at 5236.55 mass units corresponded to the CLR C1q A Pro⁵¹–Ala⁹⁷ stretch (y₄₇ ion) modified by two glycosylations and four hydroxylations. Its corresponding deglycosylated form was detected at 4912.44 mass units. The complementary sequence Glu¹–Glu⁵⁰ (b₅₀ ion), bearing three glycosylations and four hydroxylations, was detected at 6173.89 mass units. Once again, its deglycosylated form was detected at 5849.78 mass units. The b₅₀ ion revealed glycosylation of either Lys¹⁰ or Lys¹¹, a modification that could not be detected by bottom-up analysis. Another couple of fragments was detected at 7439.59 mass units (y₆₅ ion; recalculated monoisotopic mass at 7440.59), corresponding to sequence Pro³²–Ala⁹⁷, bearing three glycosylations and six hydroxylations, and at 3969.84 mass units (b₃₂ ion) corresponding to sequence Glu¹–Glu³¹, bearing two glycosylations and two hydroxylations.

Fig. 8.

Deconvoluted and deisotoped MS/MS spectrum acquired on whole C1q A chain. Detection of complementary N- and C-terminal regions from C1q A is indicated. Inset, zoom on the loss of one sialic acid group.

Fig. 9.

MS² spectrum acquired on ionic species at about m/z 1102.41 (10+) detected during top-down MS analysis of reduced CLR. Fragmentation was performed in the linear IT, and detection of fragments was carried out in the Orbitrap cell.

Fig. 10.

Three-dimensional model of human C1q highlighting positions of unmodified lysine residues. The model (2) was assembled as described previously (50). The C1q chains are colored dark blue (A), green (B), and light blue (C). The positions of the side chains of Lys⁵⁹ A, Lys⁶¹ B, and Lys⁵⁸ C (unmodified) and of Lys⁶⁵ B (hydroxylated) are shown.