| Id |
Subject |
Object |
Predicate |
Lexical cue |
| T1 |
0-94 |
Sentence |
denotes |
Enhanced mass spectrometric mapping of the human GalNAc-type O-glycoproteome with SimpleCells. |
| T2 |
95-257 |
Sentence |
denotes |
Characterizing protein GalNAc-type O-glycosylation has long been a major challenge, and as a result, our understanding of this glycoproteome is particularly poor. |
| T3 |
258-466 |
Sentence |
denotes |
Recently, we presented a novel strategy for high throughput identification of O-GalNAc glycosites using zinc finger nuclease gene-engineered "SimpleCell" lines producing homogeneous truncated O-glycosylation. |
| T4 |
467-715 |
Sentence |
denotes |
Total lysates of cells were trypsinized and subjected to lectin affinity chromatography enrichment, followed by identification of GalNAc O-glycopeptides by nLC-MS/MS, with electron transfer dissociation employed to specify sites of O-glycosylation. |
| T5 |
716-1013 |
Sentence |
denotes |
Here, we demonstrate a substantial improvement in the SimpleCell strategy by including an additional stage of lectin affinity chromatography on secreted glycoproteins from culture media (secretome) and by incorporating pre-fractionation of affinity-enriched glycopeptides via IEF before nLC-MS/MS. |
| T6 |
1014-1192 |
Sentence |
denotes |
We applied these improvements to three human SimpleCells studied previously, and each yielded a substantial increase in the number of O-glycoproteins and O-glycosites identified. |
| T7 |
1193-1421 |
Sentence |
denotes |
We found that analysis of the secretome was an important independent factor for increasing identifications, suggesting that further substantial improvements can also be sought through analysis of subcellular organelle fractions. |
| T8 |
1422-1572 |
Sentence |
denotes |
In addition, we uncovered a substantial nonoverlapping set of O-glycoproteins and O-glycosites using an alternative protease digestion (chymotrypsin). |
| T9 |
1573-1742 |
Sentence |
denotes |
In total, the improvements led to identification of 259 glycoproteins, of which 152 (59%) were novel compared with our previous strategy using the same three cell lines. |
| T10 |
1743-1957 |
Sentence |
denotes |
With respect to individual glycosites, we identified a total of 856 sites, of which 508 (59%) were novel compared with our previous strategy; this includes four new identifications of O-GalNAc attached to tyrosine. |
| T11 |
1958-2126 |
Sentence |
denotes |
Furthermore, we uncovered ≈ 220 O-glycosites wherein the peptides were clearly identified, but the glycosites could not be unambiguously assigned to specific positions. |
| T12 |
2127-2312 |
Sentence |
denotes |
The improved strategy should greatly facilitate high throughput characterization of the human GalNAc-type O-glycoproteome as well as be applicable to analysis of other O-glycoproteomes. |
