Label-Free Discovery Array Platform for the Characterization of Glycan Binding Proteins and Glycoproteins.
The identification of carbohydrate-protein interactions is central to our understanding of the roles of cell-surface carbohydrates (the glycocalyx), fundamental for cell-recognition events. Therefore, there is a need for fast high-throughput biochemical tools to capture the complexity of these biological interactions. Here, we describe a rapid method for qualitative label-free detection of carbohydrate-protein interactions on arrays of simple synthetic glycans, more complex natural glycosaminoglycans (GAG), and lectins/carbohydrate binding proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The platform can unequivocally identify proteins that are captured from either purified or complex sample mixtures, including biofluids. Identification of proteins bound to the functionalized array is achieved by analyzing either the intact protein mass or, after on-chip proteolytic digestion, the peptide mass fingerprint and/or tandem mass spectrometry of selected peptides, which can yield highly diagnostic sequence information. The platform described here should be a valuable addition to the limited analytical toolbox that is currently available for glycomics.
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Christopher J
CJ
Gray
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Antonio
A
Sánchez-Ruíz
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Ivana
I
Šardzíková
Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool, L69 7ZB, United Kingdom.
Yassir A
YA
Ahmed
Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool, L69 7ZB, United Kingdom.
Rebecca L
RL
Miller
Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato , Col. Noria Alta S/N, Guanajuato 36050, México.
Juana E
JE
Reyes Martinez
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Edward
E
Pallister
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Kun
K
Huang
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Peter
P
Both
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Mirja
M
Hartmann
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Hannah N
HN
Roberts
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Robert
R
Šardzík
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Santanu
S
Mandal
Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool, L69 7ZB, United Kingdom.
Jerry E
JE
Turnbull
Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool, L69 7ZB, United Kingdom.
Claire E
CE
Eyers
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Sabine L
0000-0003-3974-646X
SL
Flitsch
D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000737:chemistry / Q000378:metabolism; D046228:Microarray Analysis; D008895:Milk, Human / Q000378:metabolism; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D006023:Glycoproteins; D006025:Glycosaminoglycans; D037102:Lectins; D010455:Peptides; D014357:Trypsin