Utility Of Ion-Mobility Spectrometry For Deducing Branching Of Multiply Charged Glycans & Glycopeptides In A High-Throughput Positive Ion LC-FLR-IMS-MS Workflow

Edward G. Pallister†‡* Matthew S. F. Choo†, Ian Walsh†, Jien-Nee Tai†,Shi-Jie Tay†,Yuansheng Yang†, Say-Kong Ng†, Pauline M. Rudd†, Sabine L. Flitsch‡* and Terry Nguyen-Khuong†*

†Bioprocessing Technology Institute, Agency for Science Technology and Research, Singapore

‡School of Chemistry & Manchester Institute of Biotechnology (MIB), The University of Manchester, United Kingdom

*Edward Pallister edward.pallister@postgrad.manchester.ac.uk

*Sabine Flitsch: Sabine.flitsch@machester.ac.uk

*Terry Nguyen-Khuong: Terry_Nguyen_Khuong@bti.a-star.edu.sg









Introduction

This Database provides a snapshot of data acquired during Ion Mobility Mass Specrometry Analysis of Glycans.

Please note some of the advice/guidance for using the database:

1. Please cite the database if you find it useful.

2. The Database is snapshot of the data we acquired, we have presented the best representation of the results here providing you with the real CCSD plots as we feel this is more informative than 100 % peak apexes and a big table of numbers. However as a result, the database values are not replicate averaged values and the user should take that into account. (We can share replicate data if that is of interest).

3. Some of the MS2 data contains CCSDs which are poorly resolved. This is due to intensity of the prodcut ions being too weak for a good CCSD. However, we wanted to keep the data in the analysis as the MS2 product ions maybe significant without CCSD. We therefore advise that the intensity filter bar provided in the 2D plots should be set to above ~1250 for the best experience of the CCSD data in the 2D plots. This should help remove/ignore poorly resolved CCSD data.

4. Enjoy using our database :), this is still in trail state so we appreciate feedback and discussion on how this can be improved so please get in touch !

RFMS Labelled N-Glycan IMS-MS1 Intact Analysis

Name Reducing Terminus Structure 2D MS1 Plot [M+2H]2+ CCSD [M+Na+H]2+ CCSD Mass Spectrum
RFMS-Glycan F(6)A1 RFMS Label F(6)A1 F(6)A1 2D Plot F(6)A1 [M+2H]2+ F(6)A1 [M+Na+H]2+ F(6)A1 MS1 Mass Spec
RFMS-Glycan A2 RFMS Label A2 A2 2D Plot A2 [M+2H]2+ A2 [M+Na+H]2+ A2 MS1 Mass Spec
RFMS-Glycan F(6)A2 RFMS Label F(6)A2 F(6)A2 2D Plot F(6)A2 [M+2H]2+ F(6)A2 [M+Na+H]2+ F(6)A2 MS1 Mass Spec
RFMS-Glycan M5 RFMS Label M5 M5 2D Plot M5 [M+2H]2+ M5 [M+Na+H]2+ M5 MS1 Mass Spec
RFMS-Glycan F(6)A2G1 RFMS Label F(6)A2G1 F(6)A2G1 2D Plot F(6)A2G1 [M+2H]2+ F(6)A2G1 [M+Na+H]2+ F(6)A2G1 MS1 Mass Spec
RFMS-Glycan F(6)A2G1’ RFMS Label F(6)A2G1' F(6)A2G1’ 2D Plot F(6)A2G1’ [M+2H]2+ F(6)A2G1’ [M+Na+H]2+ F(6)A2G1’ MS1 Mass Spec
RFMS-Glycan F(6)A2G2 RFMS Label F(6)A2G2 F(6)A2G2 2D Plot F(6)A2G2 [M+2H]2+ F(6)A2G2 [M+Na+H]2+ F(6)A2G2 MS1 Mass Spec
RFMS-Glycan F(6)A2G1S1 RFMS Label F(6)A2G1S1 F(6)A2G1S1 2D Plot F(6)A2G1S1 [M+2H]2+ F(6)A2G1S1 [M+Na+H]2+ F(6)A2G1S1 MS1 Mass Spec
RFMS-Glycan F(6)A2G1’S1 RFMS Label F(6)A2G1S1' F(6)A2G1’S1 2D Plot F(6)A2G1’S1 [M+2H]2+ F(6)A2G1’S1 [M+Na+H]2+ F(6)A2G1’S1 MS1 Mass Spec
RFMS-Glycan F(6)A3 RFMS Label F(6)A3 F(6)A3 2D Plot F(6)A3 [M+2H]2+ F(6)A3 [M+Na+H]2+ F(6)A3 MS1 Mass Spec
RFMS-Glycan F(6)A2B RFMS Label F(6)A2B F(6)A2B 2D Plot F(6)A2B [M+2H]2+ F(6)A2B [M+Na+H]2+ F(6)A2B MS1 Mass Spec
RFMS-Glycan F(6)A2G2S2 RFMS Label F(6)A2G2S2 F(6)A2G2S2 2D Plot F(6)A2G2S2 [M+2H]2+ F(6)A2G2S2 [M+Na+H]2+ F(6)A2G2S2 MS1 Mass Spec
RFMS-Glycan F(6)A3G1 RFMS Label F(6)A3G1 F(6)A3G1 2D Plot F(6)A3G1 [M+2H]2+ F(6)A3G1 [M+Na+H]2+ F(6)A3G1 MS1 Mass Spec
RFMS-Glycan F(6)A3G1’ RFMS Label F(6)A3G1' F(6)A3G1’ 2D Plot F(6)A3G1’ [M+2H]2+ F(6)A3G1’ [M+Na+H]2+ F(6)A3G1’ MS1 Mass Spec
RFMS-Glycan F(6)A2BG1 RFMS Label F(6)A2BG1 F(6)A2BG1 2D Plot F(6)A2BG1 [M+2H]2+ F(6)A2BG1 [M+Na+H]2+ F(6)A2BG1 MS1 Mass Spec
RFMS-Glycan F(6)A2BG1’ RFMS Label F(6)A2BG1' F(6)A2BG1’ 2D Plot F(6)A2BG1’ [M+2H]2+ F(6)A2BG1’ [M+Na+H]2+ F(6)A2BG1’ MS1 Mass Spec
RFMS-Glycan F(6)A4 RFMS Label F(6)A4 F(6)A4 2D Plot F(6)A4 [M+2H]2+ F(6)A4 [M+Na+H]2+ F(6)A4 MS1 Mass Spec
RFMS-Glycan F(6)A2G2S1 RFMS Label F(6)A2G2S1 F(6)A2G2S1 2D Plot F(6)A2G2S1 [M+2H]2+ F(6)A2G2S1 [M+Na+H]2+ F(6)A2G2S1 MS1 Mass Spec
RFMS-Glycan F(6)A2G2S1’ RFMS Label F(6)A2G2S1' F(6)A2G2S1’ 2D Plot F(6)A2G2S1’ [M+2H]2+ F(6)A2G2S1’ [M+Na+H]2+ F(6)A2G2S1’ MS1 Mass Spec

RFMS Labelled N-Glycan IMS-MS/MS Glycan Sequencing Analysis

Name Reducing Terminus Structure [M+2H]2+ MS/MS 2D CCSD [M+Na+H]2+ MS/MS 2D CCSD [M+2H]2+ MS/MS Mass Spectrum [M+Na+H]2+ MS/MS Mass Spectrum
RFMS-Glycan F(6)A2G2 RFMS Label F(6)G2 2D MS/MS CCSD Plot F(6)A2G2 [M+2H]2+ 2D MS/MS CCSD Plot F(6)A2G2 [M+Na+H]2+ [M+2H]2+ F(6)A2G2 MS/MS Mass Spec [M+H+Na]2+ F(6)A2G2 MS/MS Mass Spec
RFMS-Glycan F(6)A2G1 RFMS Label F(6)A2G1 2D MS/MS CCSD Plot F(6)A2G1 [M+2H]2+ 2D MS/MS CCSD Plot F(6)A2G1 [M+Na+H]2+ [M+2H]2+ F(6)A2G1 MS/MS Mass Spec [M+H+Na]2+ F(6)A2G1 MS/MS Mass Spec
RFMS-Glycan F(6)A2G1’ RFMS Label F(6)A2G1' 2D MS/MS CCSD Plot F(6)A2G1’ [M+2H]2+ 2D MS/MS CCSD Plot F(6)A2G1’ [M+Na+H]2+ [M+2H]2+ F(6)A2G1’ MS/MS Mass Spec [M+H+Na]2+ F(6)A2G1’ MS/MS Mass Spec
RFMS-Glycan F(6)A2B RFMS Label F(6)A2B 2D MS/MS CCSD Plot F(6)A2B [M+2H]2+ 2D MS/MS CCSD Plot F(6)A2B [M+Na+H]2+ [M+2H]2+ F(6)A2B MS/MS Mass Spec [M+H+Na]2+ F(6)A2B MS/MS Mass Spec
RFMS-Glycan F(6)A3 RFMS Label F(6)A3 2D MS/MS CCSD Plot F(6)A3 [M+2H]2+ 2D MS/MS CCSD Plot F(6)A3 [M+Na+H]2+ [M+2H]2+ F(6)A3 MS/MS Mass Spec [M+H+Na]2+ F(6)A3 MS/MS Mass Spec
RFMS-Glycan F(6)A2 RFMS Label F(6)A2 2D MS/MS CCSD Plot F(6)A2 [M+2H]2+ 2D MS/MS CCSD Plot F(6)A2 [M+Na+H]2+ [M+2H]2+ F(6)A2 MS/MS Mass Spec [M+H+Na]2+ F(6)A2 MS/MS Mass Spec
RFMS-Glycan F(6)A2G1S1 RFMS Label F(6)A2G1S1 2D MS/MS CCSD Plot F(6)A2G1S1 [M+2H]2+ 2D MS/MS CCSD Plot F(6)A2G1S1 [M+Na+H]2+ [M+2H]2+ F(6)A2G1S1 MS/MS Mass Spec [M+H+Na]2+ F(6)A2G1S1 MS/MS Mass Spec
RFMS-Glycan F(6)A2G1’S1 RFMS Label F(6)A2G1'S1 2D MS/MS CCSD Plot F(6)A2G1’S1 [M+2H]2+ 2D MS/MS CCSD Plot F(6)A2G1’S1 [M+Na+H]2+ [M+2H]2+ F(6)A2G1’S1 MS/MS Mass Spec [M+H+Na]2+ F(6)A2G1’S1 MS/MS Mass Spec
RFMS-Glycan F(6)A2G2S1 RFMS Label F(6)A2G2S1 2D MS/MS CCSD Plot F(6)A2G2S1 [M+2H]2+ 2D MS/MS CCSD Plot F(6)A2G2S1 [M+Na+H]2+ [M+2H]2+ F(6)A2G2S1 MS/MS Mass Spec [M+H+Na]2+ F(6)A2G2S1 MS/MS Mass Spec
RFMS-Glycan F(6)A2G2S1’ RFMS Label F(6)A2G2S1' 2D MS/MS CCSD Plot F(6)A2G2S1’ [M+2H]2+ 2D MS/MS CCSD Plot F(6)A2G2S1’ [M+Na+H]2+ [M+2H]2+ F(6)A2G2S1’ MS/MS Mass Spec [M+H+Na]2+ F(6)A2G2S1’ MS/MS Mass Spec
RFMS-Glycan F(6)A3G1 RFMS Label F(6)A3G1 2D MS/MS CCSD Plot F(6)A3G1 [M+2H]2+ 2D MS/MS CCSD Plot F(6)A3G1 [M+Na+H]2+ [M+2H]2+ F(6)A3G1 MS/MS Mass Spec [M+H+Na]2+ F(6)A3G1 MS/MS Mass Spec
RFMS-Glycan F(6)A3G1’ RFMS Label F(6)A3G1’ 2D MS/MS CCSD Plot F(6)A3G1’ [M+2H]2+ 2D MS/MS CCSD Plot F(6)A3G1’ [M+Na+H]2+ [M+2H]2+ F(6)A3G1’ MS/MS Mass Spec [M+H+Na]2+ F(6)A3G1’ MS/MS Mass Spec
RFMS-Glycan F(6)A2BG1 RFMS Label F(6)A2BG1 2D MS/MS CCSD Plot F(6)A2BG1 [M+2H]2+ 2D MS/MS CCSD Plot F(6)A2BG1 [M+Na+H]2+ [M+2H]2+ F(6)A2BG1 MS/MS Mass Spec [M+H+Na]2+ F(6)A2BG1 MS/MS Mass Spec
RFMS-Glycan F(6)A2BG1’ RFMS Label F(6)A2BG1’ 2D MS/MS CCSD Plot F(6)A2BG1’ [M+2H]2+ 2D MS/MS CCSD Plot F(6)A2BG1’ [M+Na+H]2+ [M+2H]2+ F(6)A2BG1’ MS/MS Mass Spec [M+H+Na]2+ F(6)A2BG1’ MS/MS Mass Spec