Title : Identification of Lubricin Mucin Glycopeptides Using CID and ETD
Abstract :
In order to analyze the STP-rich region and identify the number of glycosylation sites on synovial lubricin , we adopted a combined approach using both CID- and ETD- MS2 to identify the types of glycans attached, as well as their position
Tryptic glycopeptides of both RA and OA samples were generated both before and after partial de-glycosylation (Fig. 1B) for subsequent mass spectrometric analysis
The LC-CID/ETD- MS2 approach successfully identified 185 O-glycosylated peptides
They are presented, together with the identified O-linked glycans, glycan attachment sites , and annotation method (software or manual) of each individual glycopeptide , in Table I (and in supplemental Table S1)
Predominantly core 1 and monosialylated core 1 (NeuAcα2–3Galβ1–3GalNAcα1-) O-linked glycopeptides were identified
A small proportion of disialylated core 1 (NeuAcα2–3Galβ1–3(NeuAcα2–6)GalNAcα1-)peptides were also detected
The peptides (KPAPTTPK) identified as non-glycosylated in the previously defined mucin-like domain and in the STP-rich region (VLAKPTPK and KPAPTTPK) were also shown to be glycosylated
The glycosylation of the threonine in the KPAPTTPK repeat was also shown, as indicated by the data presented in this report (supplemental Table S1)
In addition to core 1, a small proportion of core 2 (Galβ1–3(Galβ1–4GlcNAcβ1–6)GalNAcα1-) and monosialylated core 2 (e.g. NeuAcα2–3(Galβ1–3(Galβ1–4GlcNAcβ1–6)GalNAcα1-))glycopeptides were also identified
These findings are consistent with the O-linked glycans identified in our and others' previous studies (7, 26, 38)
The CID- MS2 approach effectively identified the nature of the glycans attached to lubricin
CID- MS2 spectra of four different O-linked isoforms of the same amino acid sequence (EPAPTTPK) located in the STP-rich region are presented in Figs. 2A–2D
The spectrum of the [M+2H]2+ ions at m/z 603.3 (Galβ1–3GalNAcα1-O-[EPAPTTPK]) resulted in fragmentation of the glycan component into y-type ions (Domon and Costello nomenclature) and b-type ions corresponding to [HexNAc+H]+ and [HexHexNAc+H]+ oxonium ions at m/z 204 and 366, respectively (Fig. 2A)
This corresponded to a glycopeptide with a core 1 glycan at one of the Thr residues
The spectrum showed the neutral loss of Hexresidue (m/z 1043.6), which was followed by a loss of HexNAcresidue (m/z 840.3), establishing the glycan sequence as Hex-HexNAc
However, the CID- MS2 spectrum did not show which of the Thr residues was glycosylated
The sialylated version of this glycopeptide was also identified (Fig. 2B)
The presence of an oxonium ion at m/z 292 (sialic acid, NeuAc) in the CID- MS2 spectrum of the [M+2H]2+ ions at m/z 748.8 (NeuAc-Hex-HexNAc-O-[EPAPTTPK]) showed that this glycopeptide can also be sialylated (Fig. 2B)
The loss of a NeuAcresidue (m/z 1205.3) followed by the loss of Hex (m/z 1043.3) and finally the loss of HexNAc (m/z 840.5) indicated a NeuAc-Hex-HexNAc- structure attached to a Thr residue in the peptide sequence (Fig. 2B)
This, together with previous O-glycan analysis, suggested that the attached structure was NeuAcα2–3Galβ1–3GalNAcα1-O-ThrThr
The CID- MS2 spectra allowed the identification of isomeric glycopeptides , showing differences in the number of glycosylation sites and glycan sequences
The presence of diagnostic ions at m/z 407 [HexNAc2\+H]\+ nd 569 [Hex(HexNAc)HexNAc\+H]\+ Fig. 2D) were used to differentiate a single substituted core 2 O-glycan from a doubly substituted core 1 O-glycan on both Thr residues in EPAPTTPK repeats
These results indicated that there were heavily glycosylated regions of the STP-rich region , such as the doubly glycosylated EPAPTTPK repeat shown in Fig. 2C
The STP region also displayed more complex O-glycosylation such as the core 2 structure shown in Fig. 2D
ETD- MS2 analysis was used for the identification of glycosylation sites within the STP-rich region , particularly for the identification of non-consensus repeats
Generally, the ETD- MS2 highly charged glycopeptide precursor ions fragmented efficiently, allowing the site of glycosylation to be further narrowed down, in most cases to single amino acid residues (Table I and supplemental Table S1)
All ETD- MS2 spectra were manually annotated for verification of the location of uniquely modified Ser/Thr residues in order to remove all possible ambiguity
The ETD- MS2 spectrum of the [M+3H]3+ ions at m/z 673.3 glycopeptide (K972ITTLKTTTLAPK985V) allowed the identification of the glycan-modified Thr residues within the peptide sequence
The ETD- MS2 spectrum, [M+3H]3+ ions at m/z 673.3 (Fig. 3A), displayed the z10+1-, z9+1-, and c7-ions at m/z 1788.6, 1687.8, and 1506.6, respectively, indicating that Thr974, Thr975, and Thr980 were unmodified
However, the c6-ion at m/z 1040.4 was observed with the addition of a Hex-HexNAcresidue (365 Da), indicating that the Thr979 was modified with an unsialylated core 1 structure
The c5-ion at m/z 574.4 showed that the adjacent Thr978 was modified with a second Hex-HexNAcresidue (Fig. 3A)
The z7-ion at m/z 1444.7 denoted that the two core 1 glycans (Hex-HexNAc) were still intact and attached to the peptide
However, the z5+1-ion at m/z 513.2 confirmed that both Thr978 and Thr979 were core 1 modified, as it was the loss of two threonines and two Hex-HexNAc- units
Lubricin displayed macroheterogeneity as shown by the identification of an isomeric glycopeptide (Fig. 3B)
The ETD- MS2 spectrum of the [M+3H]3+ ions at m/z 673.3 indicated the same peptide sequence (K972ITTLKTTTLAPK985V) with two core 1 glycans on separate threonine residues (Fig. 3B)
The c7-ion at m/z 1141.6 indicated the loss of the Hex-HexNAcThr980
The c6-ion at m/z 1040.6 was the loss of unmodified Thr979
The Thr978 was modified with the second Hex-HexNAcresidue identified by the c5-ion at m/z 574.5, the loss of Thr978 with a HexHexNAc
The c5-ion at m/z 574.5, a peptide with two core 1 glycans, indicated that Thr974 and Thr975 were unmodified
The ETD- MS2 spectra of the isomeric [M+3H]3+ ion at m/z 673.3 (Figs. 3A and 3B) revealed the site occupancy within the glycopeptide K972ITTLKTTTLAPK985V of SF lubricin
Overall, the dual fragmentation approach identified 185 lubricin glycopeptides , primarily from the STP-rich region
This allowed us to characterize 168 glycosylation sites , predominantly in the STP-rich region (aa 232–1056), covering 71% of the Ser/Thr in this STP-rich region
This, together with the identified non-glycosylated Ser/Thr (mainly in the N and C termini ), covered 72% of the Ser/Thr (266 out of 370 Ser/Thr were identified) in the entire protein sequence
The Ser/Thr coverage provided one of the most extensive O-glycosylation maps of a mucin-type protein (supplemental Fig
S2 and supplemental Table S1)
The identified glycosylated and non-glycosylated Ser/Thr (both in the N and C termini and in the STP-rich region ) are shown in supplemental Fig
S2
In addition to the Ser/Thr coverage, the mass spectrometric approach covered 82% of the entire protein sequence , and the coverages for the N terminus (aa 1–231), STP-rich region (aa 232–1056), and C terminus (aa 1057–1404) were 79%, 80%, and 85%, respectively