Novel cell line development strategy for monoclonal antibody manufacturing using translational enhancing technology.

Chinese hamster ovary (CHO) cells are widely used for constructing expression systems to produce therapeutic proteins. However, the establishment of high-producer clones remains a laborious and time-consuming process, despite various progresses having been made in cell line development. We previously developed a new strategy for screening high monoclonal antibody (mAb)-producing cells using flow cytometry (FCM). We also reported that p180 and SF3b4 play key roles in active translation on the endoplasmic reticulum, and that the productivity of secreted alkaline phosphatase was enhanced by the overexpression of p180 and SF3b4. Here, we attempted to apply the translational enhancing technology to high mAb-producing cells obtained after high-producer cell sorting. A high mAb-producing CHO clone, L003, which showed an mAb production level of >3 g/L in fed-batch culture, was established from a high mAb-producing cell pool fractionated by FCM. Clones generated by the overexpression of p180 and SF3b4 in L003 cells were evaluated by fed-batch culture. The specific productivity of clones overexpressing these two factors was ∼3.1-fold higher than that of parental L003 cells in the early phase of the culture period. Furthermore, the final mAb concentration was increased to 9.5 g/L during 17 days of fed-batch culture after optimizing the medium and culture process. These results indicate that the overexpression of p180 and SF3b4 would be promising for establishing high-producer cell lines applicable to industrial production.

An influenza HA stalk reactive polymeric IgA antibody exhibits anti-viral function regulated by binary interaction between HA and the antibody.

IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies (SIgAs), play an important role in preventing influenza virus infection. A recent study reported that anti-hemagglutinin (HA) head-targeting antibodies increase anti-viral functions such as hemagglutination inhibition (HI) and virus neutralization (NT), in addition to HA binding activity (reactivity) via IgA polymerization. However, the functional properties of anti-viral IgA antibodies with mechanisms of action distinct from those of anti-HA head-targeting antibodies remain elusive. Here, we characterized the functional properties of IgG, monomeric IgA, and polymeric IgA anti-HA stalk-binding clones F11 and FI6, and B12 (a low affinity anti-HA stalk clone), as well as Fab-deficient (ΔFab) IgA antibodies. We found that IgA polymerization impacts the functional properties of anti-HA stalk antibodies. Unlike anti-HA head antibodies, the anti-viral functions of anti-HA stalk antibodies were not simply enhanced by IgA polymerization. The data suggest that two modes of binding (Fab paratope-mediated binding to the HA stalk, and IgA Fc glycan-mediated binding to the HA receptor binding site (RBS)) occur during interaction between anti-stalk HA IgA antibodies and HA. In situations where Fab paratope-mediated binding to the HA stalk exceeded IgA Fc glycan-mediated binding to HA RBS, IgA polymerization increased anti-viral functions. By contrast, when IgA Fc glycan-mediated binding to the HA RBS was dominant, anti-viral activity will fall upon IgA polymerization. In summary, the results suggest that coordination between these two independent binding modules determines whether IgA polymerization has a negative or positive effect on the anti-viral functions of anti-HA stalk IgA antibodies.

The Transcription Factor HAND1 Is Involved in Cortical Bone Mass through the Regulation of Collagen Expression.

Temporal and/or spatial alteration of collagen family gene expression results in bone defects. However, how collagen expression controls bone size remains largely unknown. The basic helix-loop-helix transcription factor HAND1 is expressed in developing long bones and is involved in their morphogenesis. To understand the functional role of HAND1 and collagen in the postnatal development of long bones, we overexpressed Hand1 in the osteochondroprogenitors of model mice and found that the bone volumes of cortical bones decreased in Hand1Tg/+;Twist2-Cre mice. Continuous Hand1 expression downregulated the gene expression of type I, V, and XI collagen in the diaphyses of long bones and was associated with decreased expression of Runx2 and Sp7/Osterix, encoding transcription factors involved in the transactivation of fibril-forming collagen genes. Members of the microRNA-196 family, which target the 3' untranslated regions of COL1A1 and COL1A2, were significantly upregulated in Hand1Tg/+;Twist2-Cre mice. Mass spectrometry revealed that the expression ratios of alpha 1(XI), alpha 2(XI), and alpha 2(V) in the diaphysis increased during postnatal development in wild-type mice, which was delayed in Hand1Tg/+;Twist2-Cre mice. Our results demonstrate that HAND1 regulates bone size and morphology through osteochondroprogenitors, at least partially by suppressing postnatal expression of collagen fibrils in the cortical bones.

Heterozygous mutation of the splicing factor Sf3b4 affects development of the axial skeleton and forebrain in mouse.

BACKGROUND: Splicing factor 3B subunit 4 (SF3B4) is a causative gene of an acrofacial dysostosis, Nager syndrome. Although in vitro analyses of SF3B4 have proposed multiple noncanonical functions unrelated to splicing, less information is available based on in vivo studies using model animals. RESULTS: We performed expression and functional analyses of Sf3b4 in mice. The mouse Sf3b4 transcripts were found from two-cell stage, and were ubiquitously present during embryogenesis with high expression levels in several tissues such as forming craniofacial bones and brain. In contrast, expression of a pseudogene-like sequence of mouse Sf3b4 (Sf3b4_ps) found by in silico survey was not detected up to embryonic day 10. We generated a Sf3b4 knockout mouse using CRISPR-Cas9 system. The homozygous mutant mouse of Sf3b4 was embryonic lethal. The heterozygous mutant of Sf3b4 mouse (Sf3b4+/- ) exhibited smaller body size compared to the wild-type from postnatal to adult period, as well as homeotic posteriorization of the vertebral morphology and flattened calvaria. The flattened calvaria appears to be attributable to mild microcephaly due to a lower cell proliferation rate in the forebrain. CONCLUSIONS: Our study suggests that Sf3b4 controls anterior-posterior patterning of the axial skeleton and guarantees cell proliferation for forebrain development in mice.

Component of splicing factor SF3b plays a key role in translational control of polyribosomes on the endoplasmic reticulum.

One of the morphological hallmarks of terminally differentiated secretory cells is highly proliferated membrane of the rough endoplasmic reticulum (ER), but the molecular basis for the high rate of protein biosynthesis in these cells remains poorly documented. An important aspect of ER translational control is the molecular mechanism that supports efficient use of targeted mRNAs in polyribosomes. Here, we identify an enhancement system for ER translation promoted by p180, an integral ER membrane protein we previously reported as an essential factor for the assembly of ER polyribosomes. We provide evidence that association of target mRNAs with p180 is critical for efficient translation, and that SF3b4, an RNA-binding protein in the splicing factor SF3b, functions as a cofactor for p180 at the ER and plays a key role in enhanced translation of secretory proteins. A cis-element in the 5' untranslated region of collagen and fibronectin genes is important to increase translational efficiency in the presence of p180 and SF3b4. These data demonstrate that a unique system comprising a p180-SF3b4-mRNA complex facilitates the selective assembly of polyribosomes on the ER.

IgA tetramerization improves target breadth but not peak potency of functionality of anti-influenza virus broadly neutralizing antibody.

Mucosal immunoglobulins comprise mainly secretory IgA antibodies (SIgAs), which are the major contributor to pathogen-specific immune responses in mucosal tissues. These SIgAs are highly heterogeneous in terms of their quaternary structure. A recent report shows that the polymerization status of SIgA defines their functionality in the human upper respiratory mucosa. Higher order polymerization of SIgA (i.e., tetramers) leads to a marked increase in neutralizing activity against influenza viruses. However, the precise molecular mechanisms underlying the effects of SIgA polymerization remain elusive. Here, we developed a method for generating recombinant tetrameric monoclonal SIgAs. We then compared the anti-viral activities of these tetrameric SIgAs, which possessed variable regions identical to that of a broadly neutralizing anti-influenza antibody F045-092 against influenza A viruses, with that of monomeric IgG or IgA. The tetrameric SIgA showed anti-viral inhibitory activity superior to that of other forms only when the antibody exhibits low-affinity binding to the target. By contrast, SIgA tetramerization did not substantially modify anti-viral activity against targets with high-affinity binding. Taken together, the data suggest that tetramerization of SIgA improved target breadth, but not peak potency of antiviral functions of the broadly neutralizing anti-influenza antibody. This phenomenon presumably represents one of the mechanisms by which SIgAs present in human respiratory mucosa prevent infection by antigen-drifted influenza viruses. Understanding the mechanisms involved in cross neutralization of viruses by SIgAs might facilitate the development of vaccine strategies against viral infection of mucosal tissues.

Characterization of Angiotensin-Converting Enzyme Inhibitory Activity of X-Hyp-Gly-Type Tripeptides: Importance of Collagen-Specific Prolyl Hydroxylation.

Hydroxyproline (Hyp) is a collagen-specific amino acid formed by post-translational hydroxylation of Pro residues. Various Hyp-containing oligopeptides are transported into the blood at high concentrations after oral ingestion of collagen hydrolysate. Here we investigated the angiotensin-converting enzyme (ACE) inhibitory activity of X-Hyp-Gly-type tripeptides. In an in vitro assay, ginger-degraded collagen hydrolysate enriched with X-Hyp-Gly-type tripeptides dose-dependently inhibited ACE and various synthetic X-Hyp-Gly-type tripeptides showed ACE-inhibitory activity. In particular, strong inhibition was observed for Leu-Hyp-Gly, Ile-Hyp-Gly, and Val-Hyp-Gly with IC50 values of 5.5, 9.4, and 12.8 µM, respectively. Surprisingly, substitution of Hyp with Pro dramatically decreased inhibitory activity of X-Hyp-Gly, indicating that Hyp is important for ACE inhibition. This finding was supported by molecular docking experiments using Leu-Hyp-Gly/Leu-Pro-Gly. We further demonstrated that prolyl hydroxylation significantly enhanced resistance to enzymatic degradation by incubation with mouse plasma. The strong ACE-inhibitory activity and high stability of X-Hyp-Gly-type tripeptides highlight their potential for hypertension control.

IgA polymerization contributes to efficient virus neutralization on human upper respiratory mucosa after intranasal inactivated influenza vaccine administration.

Unlike the current injectable influenza vaccines, intranasally administered influenza vaccines induce influenza virus-specific IgA antibodies in the local respiratory mucosa as well as IgG antibodies in the systemic circulation. Our previous study showed that after five volunteers underwent intranasal administration with inactivated H3N2 or H5N1 vaccines, their IgA antibodies on the upper respiratory tract were present as monomers, dimers, and multimers (trimers and tetramers). Moreover, the multimers associated with the highest virus neutralizing activity. However, it has remained elusive whether a more practical intranasal vaccination strategy could induce the high-performance IgA multimers in the nasal mucosa. In the present study, volunteers were administered with two doses of the intranasal trivalent whole-virus inactivated influenza vaccine and showed that in nasal wash samples the amount of multimeric IgA correlated positively with virus neutralizing titers, indicating that the multimeric IgA antibodies play an important role in the antiviral activity at the nasal mucosa. Surface plasmon resonance analysis of the binding dynamics of nasal wash derived IgA monomers, dimers, and multimers against recombinant trimeric influenza virus HA showed that sample fractions containing IgA multimers dissociated from HA less well than sample fractions without IgA multimers. Thus, IgA multimers may "stick" to the antigen more tightly than the other structures. In summary, intranasal administration of two doses of multivalent inactivated influenza vaccines induced multimeric IgA. Multimerization of mucosal IgA antibodies conferred higher neutralizing activity against viruses in the nasal mucosa, possibly by increasing their cohesion to virus antigens.

Identification of Collagen-Derived Hydroxyproline (Hyp)-Containing Cyclic Dipeptides with High Oral Bioavailability: Efficient Formation of Cyclo(X-Hyp) from X-Hyp-Gly-Type Tripeptides by Heating.

Cyclic dipeptides (2,5-diketopiperazines) are present in a variety of foods and are reported to demonstrate antioxidant, antidepressant, and other beneficial effects. We recently developed a novel collagen hydrolysate characterized by a high content of X-hydroxyproline (Hyp)-Gly-type tripeptides using ginger protease. In the present study, we found that, through heating, X-Hyp-Gly can be easily converted into Hyp-containing cyclic dipeptides. After heating for 3 h at 85 °C and pH 4.8, Ala-Hyp-Gly was almost completely cyclized to cyclo(Ala-Hyp), in contrast to a slight cyclization of Ala-Hyp. The contents of cyclo(Ala-Hyp) and cyclo(Leu-Hyp) reached 0.5-1% (w/w) each in the ginger-degraded collagen hydrolysate under the heating conditions. Oral administration experiments using mice revealed that cyclo(Ala-Hyp) and cyclo(Leu-Hyp) were absorbed into the blood at markedly higher efficiencies compared to collagenous oligopeptides, including Pro-Hyp. The high productivity and oral bioavailability of the collagen-specific cyclic dipeptides suggest significant health benefits of the heat-treated ginger-degraded collagen hydrolysate.

Hydroxyhomocitrulline Is a Collagen-Specific Carbamylation Mark that Affects Cross-link Formation.

Carbamylation is a non-enzymatic post-translational modification that physiologically occurs during aging and is a risk factor for various diseases. The most common product of carbamylation is homocitrulline (HCit), where a lysine (Lys) amino group has reacted with urea-derived cyanate. HCit has recently been detected in collagen; however, given that 15%-90% of total Lys in collagen is hydroxylated, it is unclear how hydroxylation affects collagen carbamylation. Here, we identified a collagen-specific carbamylation product, hydroxyhomocitrulline (HHCit), and showed that high levels of HHCit are correlated with age in rat tissue collagen and in vivo carbamylation in mice, as well as with the decline of kidney function in the serum of dialysis patients. Proteomic analysis of the carbamylated collagens identified α2(I) Lys933, a major cross-linking site, as a preferential HHCit site. Furthermore, our results suggest that hydroxylysine carbamylation affects the mechanical properties of connective tissue by competitively inhibiting collagen cross-link formation.

Production of a novel wheat gluten hydrolysate containing dipeptidyl peptidase-IV inhibitory tripeptides using ginger protease.

Wheat gluten is a Pro-rich protein complex comprising glutenins and gliadins. Previous studies have reported that oral intake of enzymatic hydrolysates of gluten has beneficial effects, such as suppression of muscle injury and improvement of hepatitis. Here, we utilized ginger protease that preferentially cleaves peptide bonds with Pro at the P2 position to produce a novel type of wheat gluten hydrolysate. Ginger protease efficiently hydrolyzed gluten, particularly under weak acidic conditions, to peptides with an average molecular weight of <600 Da. In addition, the gluten hydrolysate contained substantial amounts of tripeptides, including Gln-Pro-Gln, Gln-Pro-Gly, Gln-Pro-Phe, Leu-Pro-Gln, and Ser-Pro-Gln (e.g. 40.7 mg/g at pH 5.2). These gluten-derived tripeptides showed high inhibitory activity on dipeptidyl peptidase-IV with IC50 values of 79.8, 70.9, 71.7, 56.7, and 78.9 µM, respectively, suggesting that the novel gluten hydrolysate prepared using ginger protease can be used as a functional food for patients with type 2 diabetes.

Efficient Absorption of X-Hydroxyproline (Hyp)-Gly after Oral Administration of a Novel Gelatin Hydrolysate Prepared Using Ginger Protease.

Recent studies have reported that oral intake of gelatin hydrolysate has various beneficial effects, such as reduction of joint pain and lowering of blood sugar levels. In this study, we produced a novel gelatin hydrolysate using a cysteine-type ginger protease having unique substrate specificity with preferential peptide cleavage with Pro at the P2 position. Substantial amounts of X-hydroxyproline (Hyp)-Gly-type tripeptides were generated up to 2.5% (w/w) concomitantly with Gly-Pro-Y-type tripeptides (5%; w/w) using ginger powder. The in vivo absorption of the ginger-degraded gelatin hydrolysate was estimated using mice. The plasma levels of collagen-derived oligopeptides, especially X-Hyp-Gly, were significantly high (e.g., 2.3-fold for Glu-Hyp-Gly, p < 0.05) compared with those of the control gelatin hydrolysate, which was prepared using gastrointestinal proteases and did not contain detectable X-Hyp-Gly. This study demonstrated that orally administered X-Hyp-Gly was effectively absorbed into the blood, probably due to the high protease resistance of this type of tripeptide.

Developmental Stage-dependent Regulation of Prolyl 3-Hydroxylation in Tendon Type I Collagen.

3-Hydroxyproline (3-Hyp), which is unique to collagen, is a fairly rare post-translational modification. Recent studies have suggested a function of prolyl 3-hydroxylation in fibril assembly and its relationships with certain disorders, including recessive osteogenesis imperfecta and high myopia. However, no direct evidence for the physiological and pathological roles of 3-Hyp has been presented. In this study, we first estimated the overall alterations in prolyl hydroxylation in collagens purified from skin, bone, and tail tendon of 0.5-18-month-old rats by LC-MS analysis with stable isotope-labeled collagen, which was recently developed as an internal standard for highly accurate collagen analyses. 3-Hyp was found to significantly increase in tendon collagen until 3 months after birth and then remain constant, whereas increased prolyl 3-hydroxylation was not observed in skin and bone collagen. Site-specific analysis further revealed that 3-Hyp was increased in tendon type I collagen in a specific sequence region, including a previously known modification site at Pro(707) and newly identified sites at Pro(716) and Pro(719), at the early ages. The site-specific alterations in prolyl 3-hydroxylation with aging were also observed in bovine Achilles tendon. We postulate that significant increases in 3-Hyp at the consecutive modification sites are correlated with tissue development in tendon. The present findings suggest that prolyl 3-hydroxylation incrementally regulates collagen fibril diameter in tendon.

Highly accurate quantification of hydroxyproline-containing peptides in blood using a protease digest of stable isotope-labeled collagen.

Collagen-derived hydroxyproline (Hyp)-containing dipeptides and tripeptides, which are known to possess physiological functions, appear in blood at high concentrations after oral ingestion of gelatin hydrolysate. However, highly accurate and sensitive quantification of the Hyp-containing peptides in blood has been challenging because of the analytical interference from numerous other blood components. We recently developed a stable isotope-labeled collagen named "SI-collagen" that can be used as an internal standard in various types of collagen analyses employing liquid chromatography-mass spectrometry (LC-MS). Here we prepared stable isotope-labeled Hyp-containing peptides from SI-collagen using trypsin/chymotrypsin and plasma proteases by mimicking the protein degradation pathways in the body. With the protease digest of SI-collagen used as an internal standard mixture, we achieved highly accurate simultaneous quantification of Hyp and 13 Hyp-containing peptides in human blood by LC-MS. The area under the plasma concentration-time curve of Hyp-containing peptides ranged from 0.663 ± 0.022 nmol/mL·h for Pro-Hyp-Gly to 163 ± 1 nmol/mL·h for Pro-Hyp after oral ingestion of 25 g of fish gelatin hydrolysate, and the coefficient of variation of three separate measurements was <7% for each peptide except for Glu-Hyp-Gly, which was near the detection limit. Our method is useful for absorption/metabolism studies of the Hyp-containing peptides and development of functionally characterized gelatin hydrolysate.

Stable isotope-labeled collagen: a novel and versatile tool for quantitative collagen analyses using mass spectrometry.

Collagens are the most abundant proteins in animals and are involved in many physiological/pathological events. Although various methods have been used to quantify collagen and its post-translational modifications (PTMs) over the years, it is still difficult to accurately quantify type-specific collagen and minor collagen PTMs. We report a novel quantitative method targeting collagen using stable isotope-labeled collagen named "SI-collagen", which was labeled with isotopically heavy lysine, arginine, and proline in fibroblasts culture. We prepared highly labeled and purified SI-collagen for use as an internal standard in mass spectrometric analysis, particularly for a new approach using amino acid hydrolysis. Our method enabled accurate collagen analyses, including quantification of (1) type-specific collagen (types I and III in this paper), (2) total collagen, and (3) collagen PTMs by LC-MS with high sensitivity. SI-collagen is also applicable to other diverse analyses of collagen and can be a powerful tool for various studies, such as detailed investigation of collagen-related disorders.

Heterogeneity of abnormal prion protein (PrP(Sc)) in murine scrapie prions determined by PrP(Sc)-specific monoclonal antibodies.

In prion diseases, abnormal prion protein (PrP(Sc)) is considered as the main component of the infectious agent. Delineation of PrP(Sc) conformation is expected to be a critical factor in understanding properties of prions. However, practical methods to differentiate between conformers of PrP(Sc) are inadequate. Here, we used two PrP(Sc)-specific monoclonal antibodies (mAbs), 3B7 and 3H6, and found that mAb 3H6 detected a limited portion of PrP(Sc) in five mice-adapted prion strains. The quantity of mAb 3H6-precipitated PrP(Sc) was significantly lesser in 22L compared to other strains. This result provides a direct evidence of the conformational heterogeneity of PrP(Sc) within the prion strains. Conformation-specific probes, like these mAbs, have the potential to be powerful tools for investigating conformational variations in PrP(Sc).

Different antigenicities of the N-terminal region of cellular and scrapie prion proteins.

Limited information is available about conformational differences between the abnormal isoform of prion protein (PrP(Sc) ) and cellular prion protein (PrP(C) ) under native conditions. To clarify conformational differences between these two isoforms, PrP-deficient mice were immunized with brain homogenates of normal and scrapie-infected animals. All mice generated anti-PrP antibodies. Peptide array analysis of these serum samples revealed a distinctive epitope of PrP(Sc) consisting of QGSPGGN (PrP41-47) at the N-terminus. This study demonstrated a conformational dissimilarity at the N-terminus between PrP(Sc) and PrP(C) , a finding that may provide novel information about conformational features of PrP(Sc) .

Site-specific quantitative analysis of overglycosylation of collagen in osteogenesis imperfecta using hydrazide chemistry and SILAC.

We recently developed a novel method for analysis of collagen O-glycosylations, which include galactosyl-hydroxylysine (GHL) and glucosyl-galactosyl-hydroxylysine (GGHL), using hydrazide chemistry (Taga, Y., Mol. Cell. Proteomics 2012, 11 (6), M111.010397). Here we investigated an overglycosylation model of collagen produced by cultured skin fibroblasts from osteogenesis imperfecta (OI) patients using this method. Many GHL/GGHL sites were identified in normal and OI type I collagens by LC-MS analysis after the glycopeptide purification procedure. Further, relative quantification was performed on each identified glycopeptide using stable isotope labeling by amino acids in cell culture (SILAC). Significant increases of GGHL were observed at respective glycosylation sites of type I collagen in OI, whereas an OI-specific glycosylation site was not found. These results demonstrated that the overglycosylation of type I collagen proceeds only at specific sites, resulting in accumulation of GGHL, rather than because of an increase of nonspecific glycosylation. Although the roles of collagen O-glycosylations in OI and even in normal conditions are still incompletely understood, the location of GHL/GGHL in the collagen sequence is suggested to be important for their functions.

Detection of active human cytomegalovirus by the promyelocytic leukemia body assay in cultures of PBMCs from patients undergoing hematopoietic stem cell transplantation.

A novel detection system was established previously for cells infected with the human cytomegalovirus (HCMV) in vitro that utilizes the unique IE1-dependent nuclear dispersion of promyelocytic leukemia (PML) bodies early in the HCMV replication cycle. This assay system, designated "the PML assay," makes use of the GFP-PML-expressing cell line SE/15, and allows real-time monitoring of infected cells by fluorescence microscopy without any staining procedures. A rapid and quantitative drug susceptibility testing was developed for low-titer clinical isolates propagated in fibroblasts in vitro. The present study sought to exploit the PML assay for evaluating in vivo status of HCMV without virus isolation. Progeny viruses were detected directly from peripheral blood mononuclear cells (PBMCs) infected in vivo obtained from hematopoietic stem cell transplantation recipients. The overall positivity of the PML assay tended to correlate with the levels of genomic DNA. Direct phenotypic susceptibility testing detected one ganciclovir (GCV)-resistant case among 19 samples, which was confirmed further by genomic and plaque reduction assays. However, in another patient with the sequence-proven mutant confirmed by sequencing, the progeny viruses exhibiting GCV-resistance were not detected. Studies on the isolated virus from the latter patient suggested the possibility that replication efficiency may differ between PBMCs and lesions infected in vivo, which may hamper the detection of GCV-resistant viruses by the PML assay, at least in this case. Taken together, the PML assay is sufficiently sensitive to monitor replication-competent HCMV directly from PBMCs infected in vivo, and provides a novel tool for comparing the characteristics of HCMV strains infected in vivo.

Development of a novel method for analyzing collagen O-glycosylations by hydrazide chemistry.

In recent years, glycopeptide purification by hydrazide chemistry has become popular in structural studies of glycoconjugates; however, applications of this method have been almost completely restricted to analysis of the N-glycoproteome. Here we report a novel method for analyzing O-glycosylations unique to collagen, which are attached to hydroxylysine and include galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine. We established a hydrazide chemistry-based glycopeptide purification method using (1) galactose oxidase to introduce an aldehyde into glycopeptides and (2) formic acid with heating to elute the bound glycopeptides by cleaving the hydrazone bond. This method allows not only identification of O-glycosylation sites in collagen but also concurrent discrimination of two types of carbohydrate substitutions. In bovine type I and type II collagens, galactosyl-hydroxylysine /glucosyl-galactosyl-hydroxylysine -containing peptides were specifically detected on subsequent comprehensive liquid chromatography (LC)/MS analysis, and many O-glycosylation sites, including unreported ones, were identified. The position of glycosylated hydroxylysine, which is determined by our unambiguous and simple method, could provide insight into the physiological role of the modifications.