The aim of the MIRAGE project is to improve the quality of glycomics data in the scientific literature by focusing on the development of publication and data reporting guidelines. The MIRAGE-LC module covers all aspects of instrument setup and modality of data handling and manipulation cross-linked with other MIRAGE recommendations.

Example Human IgG Standard

This section provides an example Human IgG glycan dataset reported in compliance with the MIRAGE Guidelines For each glycan the elution time and panel of sequential exoglycosidases (ABS, α2–3,6,8,9 sialidase; BTG, β1-3,4 galactosidase; BKF, α1-2,3,4,6 fucosidase) used to quantitate and confirm structural assignments is provided using a PGC-LC-MS/MS workflow.

No. Structure GlyTouCan GlycoStore RT (min) Relative % Area Exoglycosidase m/z calc m/z obs MS2
1 G59471TH GS734 29.8 0.38 ABS+BTG+BKF 759.8 759.7 Y
2 G96014TN GS778 32.4 0.45 ABS+BTG 840.8 840.8 N
3 G62880PC GS736 36.1 5.02 ABS+BTG+BKF 832.8 832.8 Y
4 G52231BF GS826 37.8 0.27 ABS 1067.4 1067.4 N
5 G39213VZ GS759 37.8 0.25 ABS+BTG+BKF 658.2 658.2 N
6 G45686NF GS637 38.1 4.67 ABS+BTG 913.8 913.8 Y
7 G53000RQ GS943 39.5 1.52 ABS+BTG 994.9 994.9 Y
8 G89993FE GS632 39.9 0.96 ABS+BTG 739.3 739.3 N
9 G50779LX GS648 39.9 0.14 ABS 1212.9 1212.9 N
10 G21393DK GS100026 40.1 0.79 ABS 1059.4 1059.4 Y
11 G66937TJ GS937 41 0.65 ABS+BTG 739.3 739.3 N
12 G15826PZ GS825 42.1 1.20 ABS 1140.4 1140.4 Y
13 G36191CD GS235 42.9 1.03 ABS+BTG 820.3 820.3 Y
14 G09629PQ GS100025 43.4 0.45 ABS 1285.9 1285.9 Y
15 G69812OF GS735 43.8 22.28 ABS+BTG+BKF 731.3 731.2 Y
16 G18609BT GS941 44.8 0.53 ABS 965.8 965.8 Y
17 G09782JM GS630 45.6 19.88 ABS+BTG 812.3 812.3 Y
18 G84467IZ GS100022 46 0.27 ABS 1111.4 1111.4 N
19 G09782JM GS630 46.3 12.64 ABS+BTG 812.3 812.3 Y
20 G33284MM GS816 47.7 2.06 ABS 957.8 957.8 Y
21 G28826SH GS731 47.9 15.81 ABS+BTG 893.3 893.3 Y
22 G62689JX GS683 49.1 7.79 ABS 1038.9 1038.9 Y
23 G83594ZB GS100017 50 0.98 ABS 1184.4 1184.4 Y

Supporting MS2

Peak lists available for download here

Glycoprotein Standard

Human IgG (Sigma)

Equipment

Column: Thermo Hypercarb PGC Column Dimensions: 3 μm, 100 mm × 180 μm (i.d.) Mass Spec: Agilent 6330 XCT Ultra Ion Trap LC: Agilent 1260 series HPLC

Properties of the Chromatographic Run

Gradient: 85 min Flow Rate: 2 uL/min Injection: 4 µl (8 µl injection loop)

Mobile Phase

A: 10 mM Ammonium bicarbonate B: 45% Acetonitrile in 10 mM Ammonium bicarbonate

Extended Notes

Gradient: (85 min) 100% A for 8 min, 5-35% B (8-53 min), 35-100% B (53-73 min), 100% B (73-78 min), 100% A (79-83 min) 2 min post run 100% A