Direct on-membrane glycoproteomic approach using MALDI-TOF mass spectrometry coupled with microdispensing of multiple enzymes.
We report a novel approach for direct on-membrane glycoproteomics by digestion of membrane-blotted glycoproteins with multiple enzymes using piezoelectric chemical inkjet printing technology and on-membrane direct MALDI-TOF mass spectrometry. With this approach, both N-linked glycan analyses and peptide mass fingerprinting of several standard glycoproteins were successfully performed using PNGase F and trypsin microscale digestions of the blotted spots on membrane from an SDS-PAGE gel. In addition, we performed a similar analysis for 2-DE separated serum glycoproteins as a demonstration of how the system could be used in human plasma glycoproteomics.
Kimura
Satoshi
S
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
Kameyama
Akihiko
A
Nakaya
Shuuichi
S
Ito
Hiromi
H
Narimatsu
Hisashi
H
D015180:Electrophoresis, Gel, Two-Dimensional; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry; D006801:Humans; D008566:Membranes / Q000737:chemistry; D008904:Miniaturization; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry*
D006023:Glycoproteins; D011134:Polysaccharides; D014357:Trypsin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase