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Heckendorf A.,PolyLC Inc. | Alpert A.,NeST Group
LC GC Europe | Year: 2011

When electrostatic effects are superimposed on hydrophilic interactions, specific functional groups can be selected (when the column has the opposite charge) or de-selected (when the column has the same charge). This can be exploited to isolate compounds, such as phosphopeptides, or to diminish the role of basic groups in solutes to enhance the selectivity for the rest of the molecule. © 2011 Advanstar Communications Inc.

Alpert A.J.,PolyLC Inc. | Petritis K.,Pacific Northwest National Laboratory | Petritis K.,Translational Genomic Research Institute | Kangas L.,Pacific Northwest National Laboratory | And 8 more authors.
Analytical Chemistry | Year: 2010

Here we demonstrate that separation of proteolytic peptides, having the same net charge and one basic residue, is affected by their specific orientation toward the stationary phase in ion-exchange chromatography. In electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with an anion-exchange material, the C-terminus of the peptides is, on average, oriented toward the stationary phase. In cation exchange, the average peptide orientation is the opposite. Data with synthetic peptides, serving as orientation probes, indicate that in tryptic/Lys-C peptides the C-terminal carboxyl group appears to be in a zwitterionic bond with the side chain of the C-terminal Lys/Arg residue. In effect, the side chain is then less basic than the N-terminus, accounting for the specific orientation of tryptic and Lys-C peptides. Analyses of larger sets of peptides, generated from lysates by either Lys-N, Lys-C, or trypsin, reveal that specific peptide orientation affects the ability of charged side chains, such as phosphate residues, to influence retention. Phosphorylated residues that are remote in the sequence from the binding site affect retention less than those that are closer. When a peptide contains multiple charged sites, then orientation is observed to be less rigid and retention tends to be governed by the peptides net charge rather than its sequence. These general observations could be of value in confirming a peptides identification and, in particular, phosphosite assignments in proteomics analyses. More generally, orientation accounts for the ability of chromatography to separate peptides of the same composition but different sequence. © 2010 American Chemical Society.

Hao P.,Nanyang Technological University | Ren Y.,Nanyang Technological University | Alpert A.J.,PolyLC Inc. | Siu K.S.,Nanyang Technological University
Molecular and Cellular Proteomics | Year: 2011

Identification of deamidated sites in proteins is commonly used for assignment of N-glycosylation sites. It is also important for assessing the role of deamidation in vivo. However, nonenzymatic deamidation occurs easily in peptides under conditions commonly used in treatment with trypsin and PNGase F. The impact on proteomic sample preparation has not yet been evaluated systematically. In addition, the 13C peaks of amidated peptides can be misassigned as monoisotopic peaks of the corresponding deamidated ones in database searches. The 19.34 mDa mass difference between them is proposed as a means for eliminating the resulting false positive identifications in large-scale proteomic analysis. We evaluated five groups of proteomic data, obtained mainly through an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC)-reverse phase (RP) chromatography sequence, and ascertained that nonenzymatic asparagine deamidation occurred to some extent on 4-9% of the peptides, resulting in the false positive identification of many N-glycosylation sites. A comprehensive investigation indicated that the chief causative factors were the mildly alkaline pH and prolonged incubations at 37 °C during proteomic sample preparation. An improved protocol is proposed featuring tryptic digestion at pH 6 and deglycosylation at pH 5, resulting in a significant decrease in nonenzymatic deamidation while conserving adequate digestion efficiency. The number of identified deamidation sites was improved significantly by increasing the sample loading amount in liquid chromatography-tandem MS. This permitted the identification of a significant number of glutamine deamidation sites, which featured sequence motifs largely different from those for asparagine deamidation: -Q-V-, -Q-L- and -Q-G- and, to a lesser extent, -Q-A- and -Q-E-. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Alpert A.J.,PolyLC Inc. | Hudecz O.,Research Institute of Molecular Pathology IMP | Hudecz O.,Austrian Academy of Sciences | Mechtler K.,Research Institute of Molecular Pathology IMP | Mechtler K.,Austrian Academy of Sciences
Analytical Chemistry | Year: 2015

Most phosphoproteomics experiments rely on prefractionation of tryptic digests before online liquid chromatography-mass spectrometry. This study compares the potential and limitations of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) and anion-exchange chromatography (AEX). At a pH higher than 5, phosphopeptides have two negative charges per residue and are well-retained in AEX. However, peptides with one or two phosphate groups are not separated from peptides with multiple Asp or Glu residues, interfering with the identification of phosphopeptides. At a pH of 2, phosphate residues have just a single negative charge but Asp and Glu are uncharged. This facilitates the separation of phosphopeptides from unmodified acidic peptides. Singly phosphorylated peptides are retained weakly under these conditions, due to electrostatic repulsion, unless hydrophilic interaction is superimposed in the ERLIC mode. Weak anion-exchange (WAX) and strong anion-exchange (SAX) columns were compared, with both peptide standards and a HeLa cell tryptic digest. The SAX column exhibited greater retention at pH 6 than did the WAX column. However, only about 60% as many phosphopeptides were identified with SAX at pH 6 than via ERLIC at pH 2. In one ERLIC run, 12 467 phosphopeptides were identified, including 4233 with more than one phosphate. We conclude that chromatography of phosphopeptides is best performed at low pH in the ERLIC mode. Under those conditions, the performances of the SAX and WAX materials were comparable. The data have been deposited with the ProteomeXchange with identifier PXD001333. © 2015 American Chemical Society.

Hao P.,Nanyang Technological University | Qian J.,Nanyang Technological University | Dutta B.,Nanyang Technological University | Cheow E.S.H.,Nanyang Technological University | And 5 more authors.
Journal of Proteome Research | Year: 2012

Deamidation of asparaginyl residues in proteins produces a mixture of asparaginyl, n-aspartyl, and isoaspartyl residues, which affects the proteins structure, function, and stability. Thus, it is important to identify and quantify the products to evaluate the effects in biological systems. It is still a challenging task to distinguish between the n-Asp and isoAsp deamidation products in a proteome-wide analysis because of their similar physicochemical properties. The quantification of the isomeric deamidated peptides is also rather difficult because of their coelution/poor separation in reverse-phase liquid chromatography (RPLC). We here propose a RP-ERLIC-MS/MS approach for separating and quantifying on a proteome-wide scale the three products related to deamidation of the same peptide. The key to the method is the use of RPLC in the first dimensional separation and ERLIC (electrostatic repulsion-hydrophilic interaction chromatography) in the second, with direct online coupling to tandem MS. The coelution of the three deamidation-related peptides in RPLC is then an asset, as they are collected in the same fraction. They are then separated and identified in the second dimension with ERLIC, which separates peptides on the basis of both pI and GRAVY values. The coelution of the three products in RPLC and their efficient separation in ERLIC were validated using synthetic peptides, and the performance of ERLIC-MS/MS was tested using peptide mixtures from two proteins. Applying this sequence to rat liver tissue, we identified 302 unique N-deamidated peptides, of which 20 were identified via all three deamidation-related products and 70 of which were identified via two of them. © 2012 American Chemical Society.

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