Table I. Summary of analytical approaches and instrumentation used by the participating laboratories.
| Enzyme | Chrom. | Lab. | Instrument | MS/MS | Res/Mass Acc | Workup (Rep) | |
|---|---|---|---|---|---|---|---|
| (A) Bottom-up analysis of glycopeptides | Tryp | C18 | 1 | **AB SCIEX, QstarElite, Q-TOF | CID | 15,000; 20 ppm | 1 (2) |
| 2(a) | **Bruker, AmazonETD, Ion Trap | CID | 5,000/160 ppm | 2 | |||
| 3 | Thermo, LTQ-FT Ultra | CID | 25,000 | 1 (2) | |||
| 4 | Thermo, LTQ-FT Ultra | SID | 200,000/0.2 | 1 (10) | |||
| 5 | Thermo, LTQ Orbitrap XL | CID/HCD | 60,000 | 1 (4) | |||
| 6 | Thermo, LTQ Orbitrap Velos | CID/HCD | 15,000/<5 ppm | 3 | |||
| 7 | Thermo, LTQ Orbitrap Velos | CID/HCD | 60,000/<3 ppm | 1 (5) | |||
| 8 | Thermo, LTQ Orbitrap Elite | HCD/ETD | 60,000/<10 ppm | 2 | |||
| PGC | 9(a) | Bruker, HCT 3D, Ion Trap | CID | 5,000/0.1–0.3 Da | 1 (1) | ||
| CE | 10 | Bruker, Maxis Quad, Q-TOF | CID | 40,000/1 mDa | 1 (1) | ||
| Chym | C18 | 11 | Thermo, LTQ-FT | CID | 100,000/10 ppm | 1 (2) | |
| 12 | AB SCIEX, Qstar Elite, Q-TOF | CID | 12,000/15 ppm | 3 | |||
| Tryp-Chym | 13 | Thermo, LTQ Orbitrap XL | CID | 30,000/5 ppm | 2 | ||
| ArgC | 14 | *Waters, QTOF Premier, Q-TOF | CID | 10,000/30 ppm | 1 (1) | ||
| 19(a)1 | Bruker, AmazonETD, Ion Trap | CID | 7,000/0.5 Da | 2 (3) | |||
| ArgC-Tryp | 15 | Thermo, LTQ | - | - | 1 (2) | ||
| LysC | C8 | 16 | Bruker, Maxis 4G, Q-TOF | CID | 45,000/<0.02 Da | 1 (1) | |
| C18 | 18(a)1 | Thermo, Orbitrap Elite | ETD/HCD | 240,000/<5 ppm | - | ||
| Lysyl Endo | C30 | 17 | ABI Voyager DEPro MALDI-TOF | - | 480/0.5 Da | - | |
| (B) Top-down | - | PLRP-S | 18(b) | Thermo, Orbitrap Elite | ETD/HCD | 240,000/<10 ppm | 2 |
| RP-4H | 19(b) | Bruker, Maxis 4G, Q-TOF | - | 40,000/10 ppm | 1 (2) | ||
| C8 | 20 | Bruker, Maxis, Q-TOF | - | 40,000/<0.2 Da | 1 (1) | ||
| CE | 21 | Bruker, micrOTOF-Q | - | 15,000/1 Da | 1 (1) | ||
| (C) Analysis of PNGAse F released glycans | PNGase F, Red | PGC | 9(b) | Agilent, MSD XCT 3D, Ion Trap | CID | 5,000/<0.3 Da | 1 |
| PNGase F, Red | PGC | 2(b) | Bruker, AmazonETD, Ion Trap | CID | >2,000/50 ppm | 2 | |
| PNGase F | PGC | 22 | Agilent, TOF 4224 | - | 10,000/<2 ppm | 2 (2) | |
| PNGase F Permet | - | 23 | Perspective Biosystem Voyager DE-RF, MALDI TOF | - | 10,000/<0.2 Da | 1 (3) | |
| PNGase F and other enzymes*** | HPAEC | 24 | Pulsed Amperometric Detection | - | Identification via standards | 1 (2) |
* Glycopeptides were enriched using a sialic acid capture-and-release protocol [27] where sialic acid is a prerequisite for the enrichment but is also removed during the procedure.
** ZIC-HILIC or HILIC cleanup.
*** Other enzymes used were α2–3 sialidase, non-specific neuraminidase, Neu5Ac.
1 Laboratory 18 and 19 carried out a bottom-up approach to confirm qualitative results obtained with their top-down approach. No quantitative data were submitted.