Enhanced Separation and Characterization of Deamidated Peptides with RP-ERLIC-Based Multidimensional Chromatography Coupled with Tandem Mass Spectrometry

Abstract

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.

Proteomic Study of Degenerative Protein Modifications in the Molecular Pathology of Neurodegeneration and Dementia

“…The detection of the DPM-modified peptide/protein is challenging because the DPMs containing peptides in the trypsin-digested protein sample usually exhibit very low stoichiometry; hence, it is very difficult to identify these from high abundant unmodified peptides during LC-MS/MS analysis. However, these DPMs containing peptides with different charges and hydrophilicities can be separated from unmodified peptides by using ion exchange column running in hydrophilic interaction liquid chromatography (HILIC) mode that facilitates the detection and identification by LC-MS/MS [35]. Moreover, the unmodified and modified peptides elute from ion exchange column in a predictable order based on their charge densities in LC-MS/MS mobile phase.…”

“…Hence, an accurate identification of DMPs and modification sites is important to understand the role of DPMs in human diseases. A comprehensive investigation including method development for accurate identification of DPMs has been performed for biomedical research [24,26,27,30,31,34,35]. Loss of synapses is one of the most significant contributors to the cognitive impairment manifest in VaD and other neurodegenerative diseases.…”

The Use of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) for Proteomics Research

“…Above that pH, the charge density of a WAX (weak anion-exchange) column decreases. 17 In a range of 65-70% ACN concentration, electrostatic repulsion is stronger than hydrophilic interaction for typical tryptic peptides, most of Simultaneous characterization of phosphoproteome and glycoproteome (37,39) Simultaneous characterization of proteome, phosphoproteome and glycoproteome in a single run (38) Study peptide deamidation RP-ERLIC-MS/MS to study peptide deamidation (22) which flow through the column unretained or elute within a short and similar timeframe, but phosphopeptides elute later. Beyond 70%, unmodified peptides become retained because of the increase in hydrophilic interaction.…”

“…[18][19][20] This order of elution is comparable in some respects to that of isoelectric focusing but is accomplished without ampholines or a complex mixture of buffering salts. The separation of peptides based on isoelectric point has been exploited for the study of protein/ peptide deamidation 21,22 as the original residue, Asn, and its deamidated products n-Asp and isoAsp have different pKa values.…”

Advances in purification and separation of posttranslationally modified proteins

“…by 2-DE) and converted isoaspartyl residues can be detected by autoradiography [206,207]. There is no specific enrichment procedure for isolating a deamidation proteome, and large-scale analyses thus depend on advances in mass spectrometry [208,209].…”