Fig 1.
MALDI-TOF spectra of total trypsin digest.
(A) Human collagen type III, (B) Human collagen type I, (C) Rat collagen type I. Peaks A–E in panel (A) and panel (B) and A-F in panel (C) are tryptic peptides of the corresponding collagens identified based on the agreement of their molecular weight (+1 ion) with that of the ‘theoretical value’ (assuming all Y-Pro as Hyp). The mass variants of 16 of each peak are labeled based on their mass differences from that of the ‘theoretical value’.
Fig 2.
The MS/MS spectra of tryptic peptide 435dgeagaqgapgpagpager of rat tail tendon α1(I) chain (lower case stands for the sequence from the genes).
Sequencing outcome from ion 840.88322+ (1680.7572+) (upper panel), ion 848.88012+ (1696.7521+) (middle panel) and ion 832.88642+ (1664.7623+) (lower panel). The hydroxylation sites are shown as . For clarity, only selected ions, those most relevant to the identification of residues are labeled.
Table 1.
Peptides of rat type I collagen with mass variants of 16 (the commercial sample).
Table 2.
Peptides of type I collagen from a single rat tail tendon with mass variants of 16.
Table 3.
Peptides with mass variants of 16 in human collagen.
Fig 3.
The mapping of individual unexpected hydroxylation sites on the α chains of collagen.
Sequences of the α1(I) and α2(I) chains of human (H1A1 and H1A2, respectively), the α1(I) and the α2(I) chains of rat (R1A1 and R1A2, respectively), and the α1(III) chain of human (H3A1) were arranged by the D-periodicity according to Di Lullo et al [54]: the 4 D-periods are highlighted by a colored bar of grey, yellow, cyan, and magenta, respectively; the 0.6 D is marked by the colored bar of green. The Gly-X-Y triplets including an Ox are in grey highlight. The Py residues in the tripeptide unit of Y-Gly-X are shown in red in order to reflect the potential connection with the enzyme selectivity of C-P4H (see text). The entire segment of the three highly variable regions (N- or C-HVR, see text) with multiple Ox residues are boxed. The hydroxylated proline in the telopeptide is P*. Hydroxylysines and the Gly-Pro-Lys tripeptide where the hydroxylation could not be precisely located between the Pro-Lys residues at the C-terminus of a peptide (see text) are highlighted in yellow with Lys in green font. The oxidized methionines are in blue colored font. In all cases the PTMs observed from more than one detection/sample preparation were shown in bold font. Not sequenced regions are in faint grey. The amino acid sequence of the five collagen α chains were adapted from the UniProt database.