pr500785f_si_004.xlsx (177.07 kB)
Multidimensional Fractionation Is a Requirement for Quantitation of Golgi-Resident Glycosylation Enzymes from Cultured Human Cells
dataset
posted on 2015-02-06, 00:00 authored by Chi-Hung Lin, Jenny
H. L. Chik, Nicolle H. Packer, Mark P. MolloyGlycosylation
results from the concerted action of glycosylation
enzymes in the secretory pathway. In general, gene expression serves
as the primary control mechanism, but post-translational fine-tuning
of glycosylation enzyme functions is often necessary for efficient
synthesis of specific glycan epitopes. While the field of glycomics
has rapidly advanced, there lacks routine proteomic methods to measure
expression of specific glycosylation enzymes needed to fill the gap
between mRNA expression and the glycomic profile in a “reverse
genomics” workflow. Toward developing this workflow we enriched
Golgi membranes from two human colon cancer cell lines by sucrose
density centrifugation and further mass-based fractionation by SDS-PAGE.
We then applied mass spectrometry to demonstrate a doubling in the
number of Golgi resident proteins identified, compared to the unenriched,
low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident
glycosylation enzymes, of which 23 were glycosyltransferases, were
identified making this the largest protein database so far of Golgi
resident glycosylation enzymes experimentally identified in cultured
human cells. We developed targeted mass spectrometry assays for specific
quantitation of many of these glycosylation enzymes. Our results show
that alterations in abundance of glycosylation enzymes at the protein
level were generally consistent with the resultant glycomic profiles,
but not necessarily with the corresponding glycosyltransferase mRNA
expression as exemplified by the case of O-glycan
core 1 T synthase.