Abstract
Collagen is the major protein in the extracellular matrix and plays vital roles in tissue development and function. Collagen is also one of the most processed proteins in its biosynthesis. The most prominent post-translational modification (PTM) of collagen is the hydroxylation of Pro residues in the Y-position of the characteristic (Gly-Xaa-Yaa) repeating amino acid sequence of a collagen triple helix. Recent studies using mass spectrometry (MS) and tandem MS sequencing (MS/MS) have revealed unexpected hydroxylation of Pro residues in the X-positions (X-Hyp). The newly identified X-Hyp residues appear to be highly heterogeneous in location and percent occupancy. In order to understand the dynamic nature of the new X-Hyps and their potential impact on applications of MS and MS/MS for collagen research, we sampled four different collagen samples using standard MS and MS/MS techniques. We found considerable variations in the degree of PTMs of the same collagen from different organisms and/or tissues. The rat tail tendon type I collagen is particularly variable in terms of both over-hydroxylation of Pro in the X-position and under-hydroxylation of Pro in the Y-position. In contrast, only a few unexpected PTMs in collagens type I and type III from human placenta were observed. Some observations are not reproducible between different sequencing efforts of the same sample, presumably due to a low population and/or the unpredictable nature of the ionization process. Additionally, despite the heterogeneous preparation and sourcing, collagen samples from commercial sources do not show elevated variations in PTMs compared to samples prepared from a single tissue and/or organism. These findings will contribute to the growing body of information regarding the PTMs of collagen by MS technology, and culminate to a more comprehensive understanding of the extent and the functional roles of the PTMs of collagen.
Highlights
The high sensitivity of mass spectrometry (MS) and tandem MS sequencing (MS/MS) has led to the identification of new post-translational modifications (PTMs) in fibrillar collagen and even helped to expand the field of collagen research to include archeology for the study of ancient species [1,2,3,4,5,6,7,8,9,10]
The newly discovered PTMs that are of particular interest is the 3-hydroxyproline residues (3Hyp, or 3O) in unexpected locations, since mutations in the enzymes involved in the formation of 3Hyp have been linked to severe cases of Osteogenesis Imperfecta [13, 14]
The unpredictable nature of prolyl-3-hydroxylation has hampered the application of MS in these and other related research that rely on a precise knowledge of the genomic sequence and the PTMs of specific segments of collagen
Summary
The high sensitivity of mass spectrometry (MS) and tandem MS sequencing (MS/MS) has led to the identification of new post-translational modifications (PTMs) in fibrillar collagen and even helped to expand the field of collagen research to include archeology for the study of ancient species [1,2,3,4,5,6,7,8,9,10]. Emerging from further studies of 3Hyp is an increasingly more heterogeneous pattern in terms of number, location, and the percent occupancy of this PTM [3, 9] Such varied patterns of 3Hyp make it challenging to pin down the specific molecular interactions involving 3Hyp. At the same time, collagens have been used as biomarkers for disease detection, species identification, and investigations of the involvement of collagen in cancer metastasis, tissue remodeling, the homeostasis of the extracellular matrix, and the mineralization process of bones, to name just a few [15,16,17,18]. The unpredictable nature of prolyl-3-hydroxylation has hampered the application of MS in these and other related research that rely on a precise knowledge of the genomic sequence and the PTMs of specific segments of collagen.
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