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Report No. 004 Novel analytical methods of collagen using mass spectrometry
Report No. Novel analytical methods of collagen using mass spectrometry
004

Overview

In connective tissues, such as skin, bone, and tendon, collagen is produced and formed a triple helical structure within the cell and then extracellularly secreted. Before the triple helix formation, several of collagen-constituting amino acids are modified, including hydroxylation of proline to hydroxyproline (3Hyp and 4Hyp), hydroxylation of lysine to hydroxylysine (Hyl), and glycosylation of Hyl to its glycosides (GHL and GGHL) (Figure 1). These modifications are specific to collagen consisting of a repeating [glycine-X-Y] amino acid sequence*1, which is not found in other proteins with a few exceptions. While 4Hyp is known to contribute to stabilization of the collagen triple helix, the function of other collagen modifications in the body is still unclear. In addition, although changes in the degree of collagen modifications are found in collagen-related diseases, such as osteogenesis imperfecta and fibrosis, their biological significance remains largely unknown.
Collagen has long been studied by many analytical methods using various techniques, such as protein chemistry and radioisotopes. However, there have been problems in sensitivity and accuracy to analyze 3Hyp, GHL, and GGHL, whose content in collagen is very low, and to separately detect structurally similar 3Hyp and 4Hyp. Mass spectrometer is an analytical instrument that can selectively and sensitively measure target analytes after ionization. In recent years, its performance has been largely improved, which has greatly contributed to the research progress in various fields, including collagen research. However, analysis of the unique collagen modifications has sometimes been challenging even by mass spectrometry (MS).
To overcome those problems, we have developed novel analytical methods of collagen using MS. This report introduces (1) hydrazide method and (2) stable isotope-labeled collagen.

*1: Collagen proteins are composed of unique amino acid repeats, [-(glycine-X-Y)-(glycine-X-Y)-(glycine-X-Y)-]. Glycine is present in every third position, and the X and Y positions are frequently occupied by proline and Hyp, respectively.


Figure 1. Collagen-specific modifications.

(1) Hydrazide method

In 2003, Zhang et al. reported a method for identification of N-linked glycosylation sites using a technique called hydrazide chemistry with MS (Reference 1). In their method, peptides containing N-linked glycosylation are oxidized by sodium periodate to generate aldehydes and then specifically captured on hydrazide resin, and the purified/enriched glycopeptides are measured by MS.
We applied this methodology to analysis of collagen O-glycosylations, including GHL and GGHL, but the collagen-specific glycosylations cannot be directly analyzed by the method due to their unique structures. Therefore, we modified and optimized the method, such as by using galactose oxidase instead of sodium periodate (Figure 2), and successfully established a novel hydrazide chemistry-based method for collagen O-glycosylations, which is referred to as the “hydrazide method” (Reference 2). This method enabled highly sensitive analysis of GHL/GGHL and identification of previously unknown glycosylation sites. In addition, we elucidated the manner of overglycosylation occurred in type I collagen from patients with osteogenesis imperfecta using the method (Reference 3). Although the biological role of GHL and GGHL is still incompletely understood, we expect that the hydrazide method contributes to clarification of their function.



Figure 2. Schematic diagram of the hydrazide method (GGHL-containing peptide).
This research was originally published in Molecular & Cellular Proteomics. Taga, Y., Kusubata, M., Ogawa-Goto, K. and Hattori, S. "Development of a novel method for analyzing collagen O-glycosylations by hydrazide chemistry." Mol Cell Proteomics. 2012; 11(6): M111.010397. © the American Society for Biochemistry and Molecular Biology

(2) Stable isotope-labeled collagen

One of the issues in MS analysis is quantitative accuracy. Ionization suppression (or enhancement) caused by coexisting matrix components potentially leads to quantitative errors in MS. Therefore, we developed stable isotope-labeled collagen, named SI-collagen, which can correct any errors due to experimental procedures and ionization efficiency changes in MS by mixing it into samples as an internal standard in various collagen analyses (Reference 4). By culturing fibroblast cells with stable isotopically labeled proline, lysine, and arginine, we prepared SI-collagen in which those amino acids and their modifications are all labeled. Highly accurate analysis of collagen modifications was achieved by mixing SI-collagen with collagen samples purified from tissues and cell cultures (Figure 3). In addition, SI-collagen is applicable for quantitation of total collagen using 4Hyp as a specific indicator of collagen and selective quantitation of type I and type III collagens, which is difficult by other methods. We are now expanding application of SI-collagen; for example, a variety of Hyp-containing oligopeptides appeared in blood after oral ingestion of collagen hydrolysate can be simultaneously quantitated with high sensitivity and accuracy using SI-collagen (References 5 and 6). We believe that SI-collagen is a versatile tool for various collagen analyses and contributes to the progress of future collagen research.



Figure 3. Schematic diagram of analysis with SI-collagen.
Reprinted with permission from (Taga, Y., Kusubata, M., Ogawa-Goto, K. and Hattori, S. (2014). "Stable isotope-labeled collagen: a novel and versatile tool for quantitative collagen analyses using mass spectrometry." J Proteome Res 13(8): 3671-3678). Copyright (2014) American Chemical Society.

References

1. Zhang, H, Li XJ, Martin DB, Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol. 21, 660-666 (2003)
2. Taga Y, Kusubata M, Ogawa-Goto K, Hattori S. Development of a novel method for analyzing collagen O-glycosylations by hydrazide chemistry. Mol Cell Proteomics. 11, M111.010397 (2012)
3. Taga Y, Kusubata M, Ogawa-Goto K, Hattori S. Site-specific quantitative analysis of overglycosylation of collagen in osteogenesis imperfecta using hydrazide chemistry and SILAC. J Proteome Res. 12, 2225-2232 (2013)
4. Taga Y, Kusubata M, Ogawa-Goto K, Hattori S. Stable isotope-labeled collagen: a novel and versatile tool for quantitative collagen analyses using mass spectrometry. J Proteome Res. 13, 3671-3678 (2014)
5. Taga Y, Kusubata M, Ogawa-Goto K, Hattori S. Highly accurate quantification of hydroxyproline-containing peptides in blood using a protease digest of stable isotope-labeled collagen. J Agric Food Chem 62, 12096-12102 (2014)
6. Taga Y, Iwasaki Y, Shigemura Y, Mizuno K. Improved in vivo tracking of orally administered collagen hydrolysate using stable isotope labeling and LC-MS techniques. J Agric Food Chem. 67, 4671-4678 (2019)

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