That may have led to the loss of some hydrophilic phosphopeptides. In this study, no salts or only volatile salts were used in the loading and elution solvents so that desalting was not necessary for the ERLIC fractions, and the pH and Y-27632 concentration of organic reagents in the buffers were also optimized so that unmodified and modified peptides were all retained and fractionated simultaneously. In ERLIC fractionation, unmodified peptides with net charge of +2 in the pH range 2.6�C3.9 are repelled electrostatically by the weak anion-exchange material but are still retained on the column with high organic mobile phase through hydrophilic interaction. They can then be distributed into multiple fractions during elution through the simultaneous effect of electrostatic repulsion and a decrease in hydrophilic interaction. Most of the unmodified peptides are eluted from the column in the range of 80�C65% ACN in the gradient. Phosphopeptides and glycopeptides tend to elute after unmodified peptides due to their high hydrophilicity plus the electrostatic attraction to the column by the negatively charged phosphate or sialyl- group. In addition to this enrichment process, another obvious advantage of ERLIC over SCX is that few, if any, phosphopeptides and glycopeptides elute in the flow-through, facilitating their separations and subsequent MS analysis. As false positive discovery is always a concern for high throughput analysis, we performed a control experiment here with the sample preparation procedure unchanged except without adding PNGase F to the sample in order to estimate the extent of non-specific deamidation. We found that 72 NXS/T deamidated sites were identified both in ERLIC04 and in the control samples. The false positive glycosylation sites corresponded to 10% of the total in the final result, which is due to the non-specific deamidaton that happens spontaneously either in vivo or during the sample preparation. For SCX fractionation, a shallow gradient was used to optimize the separation of phosphopeptides and sialylated glycopeptides from unmodified peptides. As expected, most of the phosphopeptides and glycopeptides identified were eluted before unmodified peptides, some of them in the flow-through. In ERLIC fractionation, solvent A was also used as the sample solvent, and different combinations of solvent A and solvent B were used to produce different elution gradients for the optimized NSC 136476 Hedgehog inhibitor fractionation of phosphopeptides and glycopeptides without significantly affecting the separation of unmodified peptides. The retention and fractionation of unmodified peptides were successfully achieved with the solvents used here. As shown in Figure 2A, the number of proteins identified in ERLIC04 was the highest in all six fractionation methods, i.e. 2929, better than with the SCX method that is so widely used for the fractionation of unmodified peptides.