Hydroxyproline Residues Research Articles

Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission.

Plants constantly renew during their life cycle and thus require to shed senescent and damaged organs. Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA. It is unknown how expression of IDA in the abscission zone leads to HAESA activation. Here we show that IDA is sensed directly by the HAESA ectodomain. Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. A central hydroxyproline residue anchors IDA to the receptor. The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism.

Early cathepsin K degradation of type II collagen in vitro and in vivo in articular cartilage

To characterize the initial events in the cleavage of type II collagen mediated by cathepsin K and demonstrate the presence of the resulting products in human and equine articular osteoarthritic cartilage. Equine type II collagen was digested with cathepsin K and the cleavage products characterized by mass spectrometry. Anti-neoepitope antibodies were raised against the most N-terminal cleavage products and used to investigate the progress of collagen cleavage, invitro, and the presence of cathepsin K-derived products in equine and human osteoarthritic cartilage. Six cathepsin K cleavage sites distributed throughout the triple helical region were identified in equine type II collagen. Most of the cleavages occurred following a hydroxyproline residue. The most N-terminal site was within three residues of the previously identified site in bovine type II collagen. Western blotting using anti-neoepitope antibodies showed that the initial cleavages occurred at the N-terminal sites and this was followed by more extensive degradation resulting in products too small to be resolved by SDS gel electrophoresis. Immunohistochemical staining of cartilage sections from equine or human osteoarthritic joints showed staining in lesional areas which was not observed in non-arthritic sites. Cathepsin K cleaves triple helical collagen by erosion from the N-terminus and with subsequent progressive cleavages. The liberated fragments can be detected in osteoarthritic cartilage and may represent useful biomarkers for disease activity.

Post-translational modifications of plant cell wall proteins and peptides: A survey from a proteomics point of view

Plant cell wall proteins (CWPs) and peptides are important players in cell walls contributing to their assembly and their remodeling during development and in response to environmental constraints. Since the rise of proteomics technologies at the beginning of the 2000's, the knowledge of CWPs has greatly increased leading to the discovery of new CWP families and to the description of the cell wall proteomes of different organs of many plants. Conversely, cell wall peptidomics data are still lacking. In addition to the identification of CWPs and peptides by mass spectrometry (MS) and bioinformatics, proteomics has allowed to describe their post-translational modifications (PTMs). At present, the best known PTMs consist in proteolytic cleavage, N-glycosylation, hydroxylation of P residues into hydroxyproline residues (O), O-glycosylation and glypiation. In this review, the methods allowing the capture of the modified proteins based on the specific properties of their PTMs as well as the MS technologies used for their characterization are briefly described. A focus is done on proteolytic cleavage leading to protein maturation or release of signaling peptides and on O-glycosylation. Some new technologies, like top–down proteomics and terminomics, are described. They aim at a finer description of proteoforms resulting from PTMs or degradation mechanisms. This article is part of a Special Issue entitled: Plant Proteomics— a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

Arabidopsis thaliana root cell wall proteomics: Increasing the proteome coverage using a combinatorial peptide ligand library and description of unexpected Hyp in peroxidase amino acid sequences.

Plant cell walls (CWs) contain a large proportion of polysaccharides (90-95% of CW mass) and proteins (5-10%) that play major roles in CW plasticity during development and in response to environmental cues. Here, we present CW proteomics data of Arabidopsis thaliana roots. Plants were cultivated in hydroponic conditions. CW protein (CWP) extracts were prepared and analyzed in two different ways in order to enlarge the coverage of the root CW proteome: proteins were analyzed either directly or following an affinity chromatography on a combinatorial peptide ligand library (CPLL) to reduce the concentration dynamic range. Proteins were identified by LC-MS/MS and bioinformatics. Altogether, 424 proteins having predicted signal peptides have been identified (CWPs). CPLL permitted to identify low-abundant CWPs never described before, thus enlarging the coverage of the root CW proteome. The number of oxidoreductases is particularly high and includes a large collection of class III peroxidases (CIII Prxs; 38 out of the 73 A. thaliana CIII Prxs). For the first time, hydroxyproline residues were localized at conserved positions in CIII Prx amino acid sequences.

Prolidase.

Prolidase (EC.3.4.13.9) or proline dipeptidase, is one of the unique enzyme capable of degrading dipeptides, in which a proline or hydroxyproline residue is located at the C-terminal position. Prolidase has a unique function in all cell types; therefore, the mechanisms and parameters involved in prolidase activity regulation are of special interest. Could prolidase be a good biomarker in different physiologic and pathologic conditions? This is an important question. There is no consensus on the answer to this question. It is of great importance during collagen turnover, inflammation, tissue fibrosis and skeletal abnormalities.Prolidase itself without other biochemical markers may not provide information to clinicians about disease activity. So, I think it should be evaluated together with other serum biochemical markers.This review will serve to discuss many in vivo functions of prolidase, as well as level prolidase activity in diagnosis and monitoring of treatment in the various diseases (Ref. 50).

Serum Prolidase Activity as a Biomarker for Choroid Plexus Calcification.

The choroid plexus (CP) performs multiple functions such as secretion and reabsorption. CP also acts as the blood-cerebrospinal fluid barrier. Prolidase plays an important role in collagen metabolism by degrading imidodipeptides, in which proline or hydroxyproline residue is located at the C-terminal end. Serum prolidase activity (SPA) may reflect the degree of fibrosis and inflammation. Choroid plexus calcification (CPC) is considered as the physiological calcification of the brain, and CPC is diagnosed by the presence of calcification in the anatomical region on computed tomography (CT). Here, CPC and non-calcified CP were defined by Hounsfield Units (HU) values of > 150 and < 50, respectively. We aimed to measure SPA in subjects with CPC and those with non-calcified CP. This study included 89 subjects who were admitted to the neurology clinic and underwent CT: 44 subjects with CPC and 45 subjects with non-calcified CP. The neurological examination of all subjects was normal; namely, the subjects with CPC were asymptomatic. The SPA level was significantly higher in the CPC group than that in the non-calcified CP group (p < 0.002), and there was a significant positive correlation between vitamin D and SPA levels in the CPC group. In contrast, the vitamin D and parathyroid hormone levels were higher in the CPC group, but the difference was not statically significant (p > 0.05). These findings indicate that SPA is a biomarker for CPC that may be predictive of future brain disease.

Colloidal Gold--Collagen Protein Core--Shell Nanoconjugate: One-Step Biomimetic Synthesis, Layer-by-Layer Assembled Film, and Controlled Cell Growth.

The biogenic synthesis of biomolecule-gold nanoconjugates is of key importance for a broad range of biomedical applications. In this work, a one-step, green, and condition-gentle strategy is presented to synthesize stable colloidal gold-collagen core-shell nanoconjugates in an aqueous solution at room temperature, without use of any reducing agents and stabilizing agents. It is discovered that electrostatic binding between gold ions and collagen proteins and concomitant in situ reduction by hydroxyproline residues are critically responsible for the formation of the core-shell nanoconjugates. The film formed by layer-by-layer assembly of such colloidal gold-collagen nanoconjugates can notably improve the mechanical properties and promote cell adhesion, growth, and differentiation. Thus, the colloidal gold-collagen nanoconjugates synthesized by such a straightforward and clean manner, analogous to a biomineralization pathway, provide new alternatives for developing biologically based hybrid biomaterials toward a range of therapeutic and diagnostic applications.

Site-selective modification of peptides: From "customizable units" to novel α-aryl and α-alkyl glycine derivatives, and components of branched peptides.

The creation of peptide libraries by site-selective modification of a few peptide substrates would increase the efficiency of discovery processes, but still is a real synthetic challenge. The site-selective modification of small peptides at serine or threonine residues, by using a short scission-addition procedure, allows the preparation of peptides with unnatural α-aryl glycines. In a similar way, the scission of hydroxyproline residues is the key step in the production of optically pure α-alkyl glycines which are precursors or components of branched peptides. With these versatile processes, a single peptide can be transformed into a variety of peptide derivatives. The process takes place under mild conditions, and good global yields are obtained. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 104: 650-662, 2015.

Chemical Synthesis of a Glycopeptide Derived from Skp1 for Probing Protein Specific Glycosylation.

Skp1 is a cytoplasmic and nuclear protein, best known as an adaptor of the SCF family of E3-ubiquitin ligases that label proteins for their degradation. Skp1 in Dictyostelium is posttranslationally modified on a specific hydroxyproline (Hyp) residue by a pentasaccharide, which consists of a Fucα1,2-Galβ-1,3-GlcNAcα core, decorated with two α-linked Gal residues. A glycopeptide derived form Skp1 was prepared to characterize the α-galactosyltransferase (AgtA) that mediates the addition of the α-Gal moieties, and to develop antibodies suitable for tracking the trisaccharide isoform of Skp1 in cells. A strategy was developed for the synthesis of the core trisaccharide-Hyp based on the use of 2-naphthylmethyl (Nap) ethers as permanent protecting groups to allow late stage installation of the Hyp moiety. Tuning of glycosyl donor and acceptor reactivities was critical for achieving high yields and anomeric selectivities of glycosylations. The trisaccharide-Hyp moiety was employed for the preparation of the glycopeptide using microwave-assisted solid phase peptide synthesis. Enzyme kinetic studies revealed that trisaccharide-Hyp and trisaccharide-peptide are poorly recognized by AgtA, indicating the importance of context provided by the native Skp1 protein for engagement with the active site. The trisaccharide-peptide was a potent immunogen capable of generating a rabbit antiserum that was highly selective toward the trisaccharide isoform of full-length Skp1.

Selective and Bioavailable Inhibitors of Collagen Prolyl 4‐Hydroxylase

Collagen is the most abundant protein in animals. A variety of diseases are associated with the overproduction of collagen including cancer metastasis. The stability of collagen relies on post‐translational modifications that occur throughout the secretory pathway. One of the most important modifications is the hydroxylation of collagen strands by collagen prolyl 4‐hydroxylases (CP4Hs). Catalysis by CP4Hs converts proline residues to hydroxyproline residues, which are essential for the conformational stability of mature collagen strands. Importantly, CP4Hs are validated targets for the treatment of metastatic breast cancer, a significant unmet clinical need. Nonetheless, a therapy based upon this strategy has yet to emerge, primarily due to the difficulty in designing selective inhibitors for members of this enzyme family. Herein, we use a chemical approach based upon the classic 2,2′‐bipyridine (bipy) scaffold to probe the active site of human CP4H1 and understand the determinants of selectivity for this enzyme. Then, we redesign the bipy scaffold to enhance its pharmacological properties and generate potent biheteroaryl inhibitors that are selective for human CP4H1. Evaluations of these inhibitors in cell culture suggest that they are bioavailable and inhibit collagen synthesis at concentrations that do not disrupt iron homeostasis or cause general cytotoxicity. These results suggest the potential use of these inhibitors as leads for the development of antimetastatic therapeutics.This work was supported by grant R01 AR044276 (NIH).

The Self-assembly of a Mini-fibril with Axial Periodicity from a Designed Collagen-mimetic Triple Helix

In this work we describe the self-assembly of a collagen-like periodic mini-fibril from a recombinant triple helix. The triple helix, designated Col108, is expressed in Escherichia coli using an artificial gene and consists of a 378-residue triple helix domain organized into three pseudo-repeating sequence units. The peptide forms a stable triple helix with a melting temperature of 41 °C. Upon increases of pH and temperature, Col108 self-assembles in solution into smooth mini-fibrils with the cross-striated banding pattern typical of fibrillar collagens. The banding pattern is characterized by an axially repeating feature of ∼35 nm as observed by transmission electron microscopy and atomic force microscopy. Both the negatively stained and the positively stained transmission electron microscopy patterns of the Col108 mini-fibrils are consistent with a staggered arrangement of triple helices having a staggering value of 123 residues, a value closely connected to the size of one repeat sequence unit. A mechanism is proposed for the mini-fibril formation of Col108 in which the axial periodicity is instigated by the built-in sequence periodicity and stabilized by the optimized interactions between the triple helices in a 1-unit staggered arrangement. Lacking hydroxyproline residues and telopeptides, two factors implicated in the fibrillogenesis of native collagen, the Col108 mini-fibrils demonstrate that sequence features of the triple helical domain alone are sufficient to "code" for axially repeating periodicity of fibrils. To our knowledge, Col108 is the first designed triple helix to self-assemble into periodic fibrils and offers a unique opportunity to unravel the specific molecular interactions of collagen fibrillogenesis.

Identification of three potent hydroxyproline O-galactosyltransferases in Arabidopsis.

Arabinogalactan proteins (AGPs) are plant-specific extracellular glycoproteins implicated in a variety of processes during growth and development. AGP biosynthesis involves O-galactosylation of hydroxyproline (Hyp) residues followed by a stepwise elongation of the complex sugar chains. However, functionally dominant Hyp O-galactosyltransferases, such that their disruption produces phenocopies of AGP-deficient mutants, remain to be identified. Here, we purified and identified three potent Hyp O-galactosyltransferases, HPGT1, HPGT2 and HPGT3, from Arabidopsis microsomal fractions. Loss-of-function analysis indicated that approximately 90% of the endogenous Hyp O-galactosylation activity is attributable to these three enzymes. AGP14 expressed in the triple mutant migrated much faster on SDS-PAGE than when expressed in wild-type, confirming a considerable decrease in levels of glycosylation of AGPs in the mutant. Loss-of-function mutant plants exhibited a pleiotropic phenotype of longer lateral roots, longer root hairs, radial expansion of the cells in the root tip, small leaves, shorter inflorescence stems, reduced fertility and shorter siliques. Our findings provide genetic evidence that Hyp-linked arabinogalactan polysaccharide chains are critical for AGP function and clues to how arabinogalactan moieties of AGPs contribute to cell-to-cell communication during plant growth and development.

Crystal structure analysis of dipeptidyl amino peptidase from P. mexicana WO24.

Dipeptidyl aminopeptidase (DAP or DPP, EC 3.4.14) catalyses the removal of dipeptides from the amino termini of peptides and proteins. In microorganisms, we have reported the identification, purification, and characterization of DAP BI, DAP BII, DAP BIII, and DAP IV (bacterial DPP4), POP fromPseudoxanthomonas mexicanaWO24, and demonstrated that DAP BI, DAP BIII, DAP IV and POP belong to the POP family and they are classified into the clan SC, family S9 in the MEROPS database. On the basis of the enzymological data we obtained, we proposed that bacterial DAPs should be classified in a manner different from that of mammalian DPPs, except for the DAP IV. The DAP IV liberates dipeptides from the free amino terminus and has a specificity for both proline and hydroxyproline residues in the penultimate position of peptides. Here, we report the first structure of the bacterial DPP IV (P. mexicanaWO24 DAP IV) complexed with an inhibitor at 2.2 Å resolution. The subunit structure is composed of two domains, the N-terminal eight-bladed β-propeller domain and the C-terminal alpha/beta/alpha sandwich catalytic domain. These structural features are conserved with clan SC S9 family. However, the N-terminal domain contains a unique helix region that extends over the active site acting as a lid, gating substrate or product access. Based upon the structural data, as well as molecular modeling, a model suggesting that the unique helix region is conserved in some kind of bacterial DPP4s except for mammalian DPP4s and some bacterial DPP4s. Some asaccharolytic and anaerobic bacteria can be used protein or peptides as an energy source. Therefore, these bacteria secrete many types of proteases and peptidases. Especially, the elucidation of degradation mechanisms of collagen, including proline and hydroxyproline, are very important from the point of view of host tissue breakdown in pathogens. Our findings suggest that different ligand recognition mechanisms from the bacterial DPP IV to mammalian DPP4 raise the possibility of an antimicrobial development targeting DPP IV from bacteria.

Biochemical and radical-scavenging properties of sea cucumber (Stichopus vastus) collagen hydrolysates

The molecular mass distribution, amino acid composition and radical-scavenging activity of collagen hydrolysates prepared from collagen isolated from the sea cucumber Stichopus vastus were investigated. β and α1 chains of the collagen were successfully hydrolysed by trypsin. The molecular mass distribution of the hydrolysates ranged from 5 to 25 kDa, and they were rich in glycine, alanine, glutamate, proline and hydroxyproline residues. The hydrolysates exhibited excellent radical-scavenging activity. These results indicate that collagen hydrolysates from S. vastus can be used as a functional ingredient in food and nutraceutical products.

Characterisation of FUT4 and FUT6 α-(1→2)-Fucosyltransferases Reveals that Absence of Root Arabinogalactan Fucosylation Increases Arabidopsis Root Growth Salt Sensitivity

Plant type II arabinogalactan (AG) polysaccharides are attached to arabinogalactan proteins (AGPs) at hydroxyproline residues, and they are very diverse and heterogeneous structures. The AG consists of a β-(1→3)-linked galactan backbone with β-(1→6)-galactan side chains that are modified mainly with arabinose, but they may also contain glucuronic acid, rhamnose or other sugars. Here, we studied the positions of fucose substitutions in AGPs, and we investigated the functions of this fucosylation. Monosaccharide analysis of Arabidopsis leaf AGP extracts revealed a significant reduction in L-Fucose content in the fut4 mutant, but not in the fut6 mutant. In addition, Fucose was reduced in the fut4 mutant in root AGP extracts and was absent in the fut4/fut6 mutant. Curiously, in all cases reduction of fucose was accompanied with a reduction in xylose levels. The fucosylated AGP structures in leaves and roots in wild type and fut mutant plants were characterised by sequential digestion with AG specific enzymes, analysis by Polysaccharide Analysis using Carbohydrate gel Electrophoresis, and Matrix Assisted Laser Desorption/Ionisation (MALDI)-Time of Flight Mass spectrometry (MS). We found that FUT4 is solely responsible for the fucosylation of AGPs in leaves. The Arabidopsis thaliana FUT4 and FUT6 genes have been previously proposed to be non-redundant AG-specific fucosyltransferases. Unexpectedly, FUT4 and FUT6 enzymes both fucosylate the same AGP structures in roots, suggesting partial redundancy to each other. Detailed structural characterisation of root AGPs with high energy MALDI-Collision Induced Dissociation MS and NMR revealed an abundant unique AG oligosaccharide structure consisting of terminal xylose attached to fucose. The loss of this structure in fut4/fut6 mutants explains the reduction of both fucose and xylose in AGP extracts. Under salt-stress growth conditions the fut4/fut6 mutant lacking AGP fucosylation exhibited a shorter root phenotype than wild type plants, implicating fucosylation of AGPs in maintaining proper cell expansion under these conditions.

An antilisterial bacteriocin BacFL31 produced by Enterococcus faecium FL31 with a novel structure containing hydroxyproline residues

A new bacterium called FL31, which was selected for its antimicrobial activity against the pathogenic bacterium Listeria monocytogenes, was identified as Enterococcus faecium and noted to produce an antibacterial proteinaceous substance (BacFL31). The active peptide from the cell-free supernatant of E. faecium FL31 was purified in four steps and the results revealed a single band with an estimated molecular mass of approximately 3.5 kDa. The N-terminal amino acid sequence of the purified BacFL31 “GLEESXGHXGQXGPXGPXGAXGP” (X = hydroxyproline) showed the presence of six hydroxyproline residues. It displayed a bactericidal mode of action against L. monocytogenes. Its application at 400 AU/g was also noted to constitute an effective approach for preventing the contamination and growth of the pathogenic bacterium L. monocytogenes during the storage of minced beef meat at 4 °C.

The Cysteine-rich Region of Type VII Collagen Is a Cystine Knot with a New Topology

Collagens are a group of extracellular matrix proteins with essential functions for skin integrity. Anchoring fibrils are made of type VII collagen (Col7) and link different skin layers together: the basal lamina and the underlying connective tissue. Col7 has a central collagenous domain and two noncollagenous domains located at the N and C terminus (NC1 and NC2), respectively. A cysteine-rich region of hitherto unknown function is located at the transition of the NC1 domain to the collagenous domain. A synthetic model peptide of this region was investigated by CD and NMR spectroscopy. The peptide folds into a collagen triple helix, and the cysteine residues form disulfide bridges between the different strands. The eight cystine knot topologies that are characterized by exclusively intermolecular disulfide bridges have been analyzed by molecular modeling. Two cystine knots are energetically preferred; however, all eight disulfide bridge arrangements are essentially possible. This novel cystine knot is present in type IX collagen, too. The conserved motif of the cystine knot is CX3CP. The cystine knot is N-terminal to the collagen triple helix in both collagens and therefore probably impedes unfolding of the collagen triple helix from the N terminus.

The degradome and the evolution of Drosophila sex peptide as a ligand for the MIP receptor

The male sex peptide (SP) of Drosophila melanogaster has wide ranging effects on females, including rejection of courting males, increased egg production, changes to the feeding habit, increased synthesis of antimicrobial peptides and elevated locomotor activity during day-time. The peptide activates receptors in sensory neurons of the female reproductive tract and can also traverse into the hemolymph and reach the central nervous system. The SP receptor involved in rejection and egg-laying responses has been shown to be identical to the receptor for the evolutionary conserved myoinhibitory peptides (MIPs) that function as neuropeptides in both males and females. Intriguingly, MIPs cannot substitute for SP when either expressed in the male accessory glands or injected into virgin females. MIPs are linear peptides with an amidated C-terminus which protects them from cleavage by carboxypeptidases, but leaves them exposed to potential attack from aminopeptidase and endopeptidase activities. In contrast, the SP region responsible for eliciting the post-mating response is cyclic and has several hydroxyproline residues N-terminal to the disulfide bridge which is expected to protect the biological activity of SP from peptidases of the male accessory gland and seminal fluid. We now present in vitro data showing that SP is metabolically stable, whereas MIPs are much more susceptible to degradation by peptidases of the male accessory gland and the hemolymph of virgin female D. melanogaster. SP has evolved relatively recently as a MIP receptor ligand that is particularly well adapted to surviving in the hostile degradome of the male accessory gland and seminal fluid.

Abscisic Acid- and Stress-Induced Highly Proline-Rich Glycoproteins Regulate Root Growth in Rice

In the root of rice (Oryza sativa), abscisic acid (ABA) treatment, salinity, or water deficit stress induces the expression of a family of four genes, REPETITIVE PROLINE-RICH PROTEIN (RePRP). These genes encode two subclasses of novel proline-rich glycoproteins with highly repetitive PX₁PX₂ motifs, RePRP1 and RePRP2. RePRP orthologs exist only in monocotyledonous plants, and their functions are virtually unknown. Rice RePRPs are heavily glycosylated with arabinose and glucose on multiple hydroxyproline residues. They are significantly different from arabinogalactan proteins that have glycan chains composed of arabinose and galactose. Transient and stable expressions of RePRP-green fluorescent protein reveal that a fraction of this protein is localized to the plasma membrane. In rice roots, ABA treatment increases RePRP expression preferentially in the elongation zone. Overexpression of RePRP in transgenic rice reduces root cell elongation in the absence of ABA, similar to the effect of ABA on wild-type roots. Conversely, simultaneous knockdown of the expression of RePRP1 and RePRP2 reduces the root sensitivity to ABA, indicating that RePRP proteins play an essential role in ABA/stress regulation of root growth and development. Moreover, rice RePRPs specifically interact with a polysaccharide, arabinogalactan, in a dosage-dependent manner. It is suggested that RePRP1 and RePRP2 are functionally redundant suppressors of root cell expansion and probably act through interactions with cell wall components near the plasma membrane.

LC–MS/MS Identification of the O-Glycosylation and Hydroxylation of Amino Acid Residues of Collagen α-1 (II) chain from Bovine Cartilage

O-Glycosylation of collagen is a unique type of posttranslational modifications (PTMs) involving the attachment of galactose (Gal) or glucose-galactose (Glc-Gal) moieties to hydroxylysine (HyK). Also, hydroxyproline (HyP) result from the posttranslational hydroxylation of some proline residues in collagen. Here, LC-MS/MS was effectively employed to identify 23 O-glycosylation sites and a large number of HyP residues associated with bovine type II collagen α-1 chain (CO2A1). The modifications of the 23 O-glycosylation sites varied qualitatively and quantitatively. Both Gal and Glc-Gal moieties occupied 22 of the identified glycosylation sites, while K773 was observed as unmodified. A large number of HyP residues at Yaa positions of Gly-Xaa-Yaa motif were detected. HyP residues at Xaa positions of Gly-HyP-HyP, Gly-HyP-Ala, and Gly-HyP-Val motifs were also observed. Notably, HyP residue of Gly-HyP-Gln motif was detected, which has not been previously reported. Moreover, the deamidation of 8 Asn residues was identified, of which 2 Asp residues were observed at different retention times because of isomerization (Asp vs isoAsp). Partial macroheterogeneities of some CO2A1 glycosylation sites were revealed by LC-MS/MS analysis. ETD experiments revealed partial macroheterogeneities associated with K299-K308, K452-K464, K464-K470, and K857-K884 glycosylation sites. Semiquantitative data suggest that the glycosylation of hydroxylysine residues is site-specific.