Mass Spectrometry Group, NIH/National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.
J Am Soc Mass Spectrom. 2013 Jul;24(7):1072-81. doi: 10.1007/s13361-013-0624-y. Epub 2013 Apr 30.
Collagens are the most abundant glycoproteins in the body. One characteristic of this protein family is that the amino acid sequence consists of repeats of three amino acids -(X-Y-Gly)n. Within this motif, the Y residue is often 4-hydroxyproline (HyP) or 5-hydroxylysine (HyK). Glycosylation in collagen occurs at the 5-OH group in HyK in the form of two glycosides, galactosylhydroxylysine (Gal-HyK) and glucosyl galactosylhydroxylysine (GlcGal-HyK). In collision induced dissociation (CID), collagen tryptic glycopeptides exhibit unexpected gas-phase dissociation behavior compared to typical N- and O-linked glycopeptides (i.e., in addition to glycosidic bond cleavages, extensive cleavages of the amide bonds are observed). The Gal- or GlcGal- glycan modifications are largely retained on the fragment ions. These features enable unambiguous determination of the amino acid sequence of collagen glycopeptides and the location of the glycosylation site. This dissociation pattern was consistent for all analyzed collagen glycopeptides, regardless of their length or amino acid composition, collagen type or tissue. The two fragmentation pathways-amide bond and glycosidic bond cleavage-are highly competitive in collagen tryptic glycopeptides. The number of ionizing protons relative to the number of basic sites (i.e., Arg, Lys, HyK, and N-terminus) is a major driving force of the fragmentation. We present here our experimental results and employ quantum mechanics calculations to understand the factors enhancing the labile character of the amide bonds and the stability of hydroxylysine glycosides in gas phase dissociation of collagen glycopeptides.
胶原是体内最丰富的糖蛋白。这类蛋白质的一个特点是，其氨基酸序列由三个氨基酸的重复组成-(X-Y-Gly)n。在这个基序中，Y 残基通常是 4-羟脯氨酸（HyP）或 5-羟赖氨酸（HyK）。在胶原中，糖基化发生在 HyK 的 5-OH 基团上，形式为两种糖苷，半乳糖基羟赖氨酸（Gal-HyK）和葡萄糖基半乳糖基羟赖氨酸（GlcGal-HyK）。在碰撞诱导解离（CID）中，与典型的 N-和 O-连接糖肽相比，胶原酶解糖肽表现出意想不到的气相解离行为（即，除了糖苷键的断裂外，还观察到酰胺键的广泛断裂）。Gal-或 GlcGal-聚糖修饰在片段离子上基本保留。这些特征使得胶原糖肽的氨基酸序列和糖基化位点的确定变得明确。这种解离模式对于所有分析的胶原糖肽都是一致的，无论其长度、氨基酸组成、胶原类型或组织如何。酰胺键和糖苷键断裂这两种断裂途径在胶原酶解糖肽中是高度竞争的。相对于碱性位点（即 Arg、Lys、HyK 和 N-末端）的质子数是片段化的主要驱动力。我们在此展示我们的实验结果，并采用量子力学计算来理解增强酰胺键的不稳定性和羟赖氨酸糖苷在胶原糖肽气相解离中稳定性的因素。
