We have developed a simple method for the release and isolation of glycoprotein N-glycans from whole-cell lysates using less than a million cells, for subsequent implementation with mass spectrometric analysis. Cellular protein extracts prepared using SDS solubilization were sequentially treated in a membrane filter device to ultimately release glycans enzymatically using PNGase F in the volatile buffer ammonium bicarbonate. The released glycans are recovered in the filtrate following centrifugation and typically permethylated prior to mass spectrometric analysis. We call our method “filter-aided N-glycan separation” and have successfully applied it to investigate N-glycan profiles of wild-type and mutant Chinese hamster ovary cells. This method is readily multiplexed and, because of the small numbers of cells needed, is compatible with the analysis of replicate samples to assess the true nature of glycan variability in tissue culture samples.Keywords: cultured mammalian cells; filter-aided sample preparation; MALDI-MS; N-glycans; permethylation; SDS solubilization;

We have explored two divinylbenzene cross-linked polystyrene supports for use in a solid-supported N-terminal peptide tagging strategy. Resin-bound tags designed to be cleaved in a single step at the N-terminus of peptides have been devised and explored as peptide N-terminal tagging reagents (constructs) for subsequent mass spectrometric analysis. While the brominated tagging approach shows promise, the use of these specific solid supports has drawbacks, in terms of tagging reaction scale, for real applications in proteomics.Keywords: bromine; mass spectrometry; peptide labeling; proteome; solid-phase synthesis