The present strategy will facilitate such analysis by identifying proteins with both these major modifications. We have published two novel ERLIC based fractionation approaches for the simultaneous characterization of glyco- and phosphoproteomes of mouse brain membrane and the comprehensive KRX-0401 157716-52-4 profiling of rat kidney proteome. In the first one, both glycopeptides and phosphopeptides were selectively enriched due to their hydrophilic interaction and/or electrostatic interaction of the negative charged phosphoric and VE-821 1232410-49-9 sialyl groups with the stationary phase of the ERLIC column, but most unmodified peptides were excluded as flow-through since they were repelled by the stationary phase at 70% ACN at pH 2. In the second one, 90% ACN/0.1% acetic acid was used as mobile phase A so that nearly all peptides will be retained on the ERLIC column through hydrophilic and/or electrostatic interactions, and they were evenly distributed into multiple fractions based on both pI and polarity when eluted using a shallow gradient of increasing water content and decreasing pH value. However, both glycopeptides and phosphopeptides cannot be characterized effectively due to their sub-stoichiometric amounts and the ionization suppression from unmodified peptides. In this study, we further optimized the ERLIC conditions so that as many as unmodified peptides were retained and fractionated by the column when phosphopeptides and glycopeptides were enriched. Its limitation is that both the analysis of modified peptides and unmodified peptides was compromised to a certain extent compared with the two previously published approaches. However, it provided a global analysis of both unmodified peptides and modified peptides in one run, which could not be achieved with the two previously published ERLIC approaches. It was also capable of detecting partial phosphorylation and N-glycosylation with potential biological significance regarding the control of some biological processes, such as cellular component organization and transport. At the same time, it identified some proteins having both these modifications, which would facilitate the future evaluation of crosstalk between these two vital PTMs. In the future, when the present method is employed together with some quantitative methods, such as SILAC, iTRAQ or labelfree quantification methods, it will be capable of assessing the changes in protein expression and these two PTMs in one analysis, which reduces the inter-experimental variations in the quantitation. Better understanding of substoichiometric modifications may be helpful in elucidation of how some biological processes are controlled inside the cell. The Vibrio genus is ubiquitous and abundant throughout the aquatic environment. It is clear that lateral gene transfer events play a major role in the evolution and adaptation of this organism, with genetic interchange of Vibrio genes observed over a wide range of phylogenetic distances.