Electrostatic Repulsion-hydrophilic Interaction Chromatography Research Articles

Electrostatic Repulsion Hydrophilic Interaction Liquid Chromatography (ERLIC) for the Quantitative Analysis of Polyamines

Highly polar low molecular weight organic molecules are still very challenging to analyze by liquid chromatography. Yet, with the steadily increasing application of metabolomics and similar approaches in chemical analysis, separating polar compounds might be even more important. However, almost all established liquid chromatography techniques (i.e., normal and reversed phase, hydrophilic interaction liquid chromatography (HILIC), ion chromatography) struggle with either carry-over, low sensitivity, or a lack of retention. For improving these shortcomings, electrostatic repulsion hydrophilic interaction chromatography (ERLIC) might be an alternative. By combining a HILIC mobile phase, that is highly organic with a low water content, and an ion exchange column, a distinct layer system develops. When the analyte's charge is of the same direction as the stationary phase, retention and elution are determined by two antagonistic forces: electrostatic repulsion and hydrophilicity. One prominent group of challenging polar analytes are the polyamines cadaverine, putrescine, spermidine, and spermine. Carrying charges from +2 to +4 at physiological pH, these compounds are essential cell constituents and found in all living organisms. However, they are still notoriously challenging to analyze via the established liquid chromatography methods. In the present work, an ERLIC tandem mass spectrometry method has been exemplarily developed, optimized, and validated for the quantitative determination of cadaverine, putrescine, spermidine, and spermine. This method enables symmetrical peak shapes and good separation of analytes with different charges while simultaneously selectively detecting the co-eluting diamines by MS/MS. Furthermore, high linearity (R > 0.998) and sensitivity (LODs ≤ 2 ng/mL) have been proven. Thus, ERLIC may be interesting for both targeted and untargeted analysis approaches of highly charged low molecular weight organic molecules.

Counterion Optimization Dramatically Improves Selectivity for Phosphopeptides and Glycopeptides in Electrostatic Repulsion-Hydrophilic Interaction Chromatography.

A well-hydrated counterion can selectively and dramatically increase retention of a charged analyte in hydrophilic interaction chromatography. The effect is enhanced if the column is charged, as in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). This combination was exploited in proteomics for the isolation of peptides with certain post-translational modifications (PTMs). The best salt additive examined was magnesium trifluoroacetate. The well-hydrated Mg+2 ion promoted retention of peptides with functional groups that retained negative charge at low pH, while the poorly hydrated trifluoroacetate counterion tuned down the retention due to the basic residues. The result was an enhancement in selectivity ranging from 6- to 66-fold. These conditions were applied to a tryptic digest of mouse cortex. Gradient elution produced fractions enriched in peptides with phosphate, mannose-6-phosphate, and N- and O-linked glycans. The numbers of such peptides identified either equaled or exceeded the numbers afforded by the best alternative methods. This method is a productive and convenient way to isolate peptides simultaneously that contain a number of different PTMs, facilitating study of proteins with "crosstalk" modifications. The fractions from the ERLIC column were desalted prior to C-18-reversed phase liquid chromatography-tandem mass spectrometry analysis. Between 47-100% of the peptides with more than one phosphate or sialyl residue or with a mannose-6 phosphate group were not retained by a C-18 cartridge but were retained by a cartridge of porous graphitic carbon. This finding implies that the abundance of such peptides may have been significantly underestimated in some past studies.

Enrichment of Intact Glycopeptides Using Strong Anion Exchange and Electrostatic Repulsion Hydrophilic Interaction Chromatography.

Glycosylation is a biologically important and complex protein posttranslational modification. The emergence of glycoproteomic technologies to identify and characterize glycans on proteins has the potential to enable a better understanding the role of glycosylation in biology, disease states, and other areas of interest. In particular, the analysis of intact glycopeptides by mass spectrometry allows information about glycan location and composition to be ascertained. However, such analysis is often complicated by extensive glycan diversity and the low abundance of glycopeptides in a complex mixture relative to nonglycosylated peptides. Enrichment of glycopeptides from a protein enzymatic digest is an effective approach to overcome such challenges. In this chapter, we described a glycopeptide enrichment method combining strong anion exchange, electrostatic repulsion, and hydrophilic interaction chromatography (SAX-ERLIC). Following enzymatic digestion of proteins into peptides, SAX-ERLIC is performed by solid phase extraction to enrich glycopeptides from biological samples with subsequent LC-MS/MS analysis. Glycopeptide data generated using the SAX-ERLIC enrichment yields a high number of total and unique glycopeptide identifications which can be mapped back to proteins. The enrichment strategy is robust, easy to perform, and does not require cleavage of glycans prior to LC-MS/MS analysis.

Analysis of pancreatic extracellular matrix protein post-translational modifications via electrostatic repulsion-hydrophilic interaction chromatography coupled with mass spectrometry.

The pancreas is a vital organ with digestive and endocrine roles, and diseases of the pancreas affect millions of people yearly. A better understanding of the pancreas proteome and its dynamic post-translational modifications (PTMs) is necessary to engineer higher fidelity tissue analogues for use in transplantation. The extracellular matrix (ECM) has major roles in binding and signaling essential to the viability of insulin-producing islets of Langerhans. To characterize PTMs in the pancreas, native and decellularized tissues from four donors were analyzed. N-Glycosylated and phosphorylated peptides were simultaneously enriched via electrostatic repulsion-hydrophilic interaction chromatography and analyzed with mass spectrometry, maximizing PTM information from one workflow. A modified surfactant and chaotropic agent assisted sequential extraction/on-pellet digestion was used to maximize solubility of the ECM. The analysis resulted in the confident identification of 3650 proteins, including 517 N-glycoproteins and 148 phosphoproteins. We identified 214 ECM proteins, of which 99 were N-glycosylated, 18 were phosphorylated, and 9 were found to have both modifications. Collagens, a major component of the ECM, were the most highly glycosylated of the ECM proteins and several were also heavily phosphorylated, raising the possibility of structural and thus functional changes resulting from these modifications. To our knowledge, this work represents the first characterization of PTMs in pancreatic ECM proteins. This work provides a basal profile of PTMs in the healthy human pancreatic ECM, laying the foundation for future investigations to determine disease-specific changes such as in diabetes and pancreatic cancer, and potentially helping to guide the development of tissue replacement constructs. Data are available via ProteomeXchange with identifier PXD025048.

Finding the Sweet Spot in ERLIC Mobile Phase for Simultaneous Enrichment of N-Glyco and Phosphopeptides.

Simultaneous enrichment of glyco- and phosphopeptides will benefit the studies of biological processes regulated by these posttranslational modifications (PTMs). It will also reveal potential crosstalk between these two ubiquitous PTMs. Unlike custom-designed multifunctional solid phase extraction (SPE) materials, operating strong anion exchange (SAX) resin in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) mode provides a readily available strategy to analytical labs for enrichment of these PTMs for subsequent mass spectrometry (MS)-based characterization. However, the choice of mobile phase has largely relied on empirical rules from hydrophilic interaction chromatography (HILIC) or ion-exchange chromatography (IEX) without further optimization and adjustments. In this study, ten mobile phase compositions of ERLIC were systematically compared; the impact of multiple factors including organic phase proportion, ion pairing reagent, pH, and salt on the retention of glycosylated and phosphorylated peptides was evaluated. This study demonstrated good enrichment of glyco- and phosphopeptides from the nonmodified peptides in a complex tryptic digest. Moreover, the enriched glyco- and phosphopeptides elute in different fractions by orthogonal retention mechanisms of hydrophilic interaction and electrostatic interaction in ERLIC, maximizing the LC-MS identification of each PTM. The optimized mobile phase can be adapted to the ERLIC HPLC system, where the high resolution in separating multiple PTMs will benefit large-scale MS-based PTM profiling and in-depth characterization.

Profiling of Human Milk Oligosaccharides for Lewis Epitopes and Secretor Status by Electrostatic Repulsion Hydrophilic Interaction Chromatography Coupled with Negative-Ion Electrospray Tandem Mass Spectrometry.

Human milk oligosaccharides (HMOs) are one of the most abundant ingredients in breast milk, and they play a beneficial role for newborns and are important for infant health. The peripheral fucosylated sequences of HMOs, such as the histo-blood group ABH(O) and Lewis a, b, x, and y antigens, are determined by the expression of the secretor (Se) and Lewis (Le) genes in the mammary gland, and are often the recognition motifs and serve as decoy receptors for microbes. In this work, we developed a method for determination of secretor status and Lewis blood phenotype and assignment of Lewis blood-group epitopes. The method was based on electrostatic repulsion/hydrophilic interaction chromatography coupled with tandem mass spectrometry (ERLIC-MS/MS). A specifically designed stationary phase, aspartic acid-bonded silica (ABS), was used to separate the acidic and neutral HMOs by electrostatic repulsion followed by HILIC. Negative-ion electrospray MS/MS was then used for analysis of secretor status and Lewis blood phenotypes and assignment of important epitopes of HMOs from the lactating mothers by selecting a specific set of unique fragment ions.

Simultaneous Enrichment of Plasma Extracellular Vesicles and Glycoproteome for Studying Disease Biomarkers.

To detect disease at an early stage and to develop effective disease treatment therapies, reliable biomarkers of diagnosis, disease progression, and its status remain a research priority. A majority of disease pathologies are primarily associated with different subsets of cells of different tissues, discrete compartments, and areas. These subsets of cells release glycoproteins and specific extracellular vesicles (EVs) including microvesicles and exosomes that carry bioactive cargoes of proteins, nucleic acids, and metabolites. Body fluids like blood plasma are considered as a golden source of disease biomarkers since it contains glycoprotein and EVs released by almost all cell types. The contents of glycoproteome and EV cargo change with cell status, and they act as mirror of cell's intracellular events and status; hence, EVs and glycoproteins are promising disease biomarkers. However, their abundance in blood plasma remains low posing a serious technical problem in their identification and quantification. Until recently, technical advances and exhaustive research devised a technique for either enrichment of plasma glycoprotein or EVs, but no methodologies exist that can enrich and identify both plasma glycoprotein and EVs. To overcome this technical challenge, a method that can eliminate high-abundance entities without depleting disease-modifying molecules is required. Therefore, here we describe the detailed protocol of simultaneous enrichment of glycoproteins and EVs from blood plasma by prolonged ultracentrifugation coupled to electrostatic repulsion-hydrophilic interaction chromatography (PUC-ERLIC) and their identification and quantification by mass spectrometry-based proteomic technique.

A comparative study of phosphopeptide-selective techniques for a sub-proteome of a complex biological sample

Phosphorylation of proteins is important for controlling cellular signaling and cell cycle regulatory events. The process is reversible and phosphoproteins normally constitute a minor part of the global proteome in a cell. Thus, sample preparation techniques tailored for phosphoproteome studies are continuously invented and evaluated. This paper aims at evaluating the performances of the most popular techniques for phospho-enrichments in sub-proteome analysis, such as viral proteomes expressed in human cells during infection. A two-species sample of Adenovirus type 2 infected human cells was used, and in-solution digestion, strong cation exchange (SCX), and electrostatic repulsion hydrophilic interaction chromatography (ERLIC) fractionation, and subsequent enrichment by TiO2, were compared with SDS-PAGE fractionation and in-gel digestion. Evaluation was focused on phosphopeptide detection in the sub-proteome. The results showed that the SCX+TiO2 or ERLIC+TiO2 combinations had the highest enrichment efficiencies, but SDS-PAGE fractionation and in-gel digestion resulted in the highest number of identified proteins and phosphopeptides. Furthermore, the study demonstrates the usefulness of applying as many orthogonal techniques as possible in deep phosphoproteome analysis, since the overlap between approaches was low.

Enrichment Strategies in Phosphoproteomics.

The comprehensive study of the phosphoproteome is heavily dependent on appropriate enrichment strategies that are most often, but not exclusively, carried out on the peptide level. In this chapter, I give an overview of the most widely used techniques. In addition to dedicated antibodies, phosphopeptides are enriched by their selective interaction with metals in the form of chelated metal ions or metal oxides. The negative charge of the phosphate group is also exploited in a variety of chromatographic fractionation methods that include different types of ion exchange chromatography, hydrophilic interaction chromatography (HILIC), and electrostatic repulsion HILIC (ERLIC) chromatography. Selected examples from the literature will demonstrate how a combination of these techniques with current high-performance mass spectrometry enables the identification of thousands of phosphorylation sites from various sample types.

Fast and easy phosphopeptide fractionation by combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis.

Mass spectrometry-based phosphoproteomic analysis is a powerful method for gaining a global, unbiased understanding of cellular signaling. Its accuracy and comprehensiveness stands or falls with the quality and choice of the applied phosphopeptide prefractionation strategy. This protocol covers a powerful but simple and rapid strategy for phosphopeptide prefractionation. The combinatorial use of two distinct chromatographic techniques that address the inverse physicochemical properties of peptides allows for superior fractionation efficiency of multiple phosphorylated peptides. In the first step, multiphosphorylated peptides are separated according to the number of negatively charged phosphosites by electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). A subsequent strong cation exchange (SCX) step separates mostly singly phosphorylated peptides in the ERLIC flow-through according to their positive charge. The presented strategy is inexpensive and adaptable to large and small amounts of starting material, and it allows highly multiplexed sample preparation. Because of its implementation as solid-phase extraction, the entire workflow takes only 2 h to complete.

High fidelity approach for proteomic scale enrichment and identification of N-termini

A novel workflow was designed for the large-scale identification of protein N-terminal sequences. The workflow started with converting lysine to homoarginine, followed by reaction with sulfonation of N-termini by 4-sulfophenyl isothiocyanate (SPITC). Upon trypsin digestion, the SPITC modified N-terminal peptides were enriched by electrostatic repulsion hydrophilic interaction (ERLIC) chromatography in which the internal and C-terminal peptides eluted at the void volume, and the SPITC peptides were retained in the column due to the hydrophilicity and electrostatic attraction of the sulfonyl group to the stationary phase. Upon higher-energy collisional induced dissociation (HCD) SPITC peptides in ESI generated predominately y-type ion series; such simplification of spectra enables the identification of N-termini with high confidence. The appearance of b1+SPITC product ions upon HCD further boosts the confidence for N-terminal identifications. This method was applied to an Escherichia coli cell lysate, thus allowing the identification of 358 high confidence N-terminal peptides representing 274 distinct E. coli proteins as certain proteins were found to have multiple N-terminal peptides due to cleavage from various cellular enzymes. Confidence in N-terminal assignments is further heightened since 224 of the unique proteins identified from N-terminal peptides were also identified in the analysis of flow-through fractions.

Abstract B24: A phosphoproteomic comparison of oncogenic BRAF and MKK1/2 inhibition in human melanoma

Abstract The purpose of this study was to use phosphoproteomics to gain insight into the different clinical responses to BRAF inhibition, MKK1/2 inhibition, and combined BRAF/MKK inhibition. Experiments were designed to test whether BRAF inhibition may affect a distinct subset of targets that MKK1/2 inhibition does not and whether BRAF and MKK1/2 inhibitors work additively or synergistically when used in combination. To this end, we employed a SILAC-based method to quantify changes in the phosphoproteome of WM239A melanoma cells treated with a clinically relevant BRAF inhibitor (PLX4032; 10μM) or MEK1/2 inhibitor (AZD6244; 10μM) inhibitor. Phosphopeptides were enriched by titanium dioxide prior to fractionation on an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) column. In a set of three biological replicate experiments, we quantified changes at 23,986 phosphosites on 4,722 proteins. We identified 1,356 phosphosites that decreased in abundance, and 341 that increased in abundance, in response to treatment with either PLX4032 or AZD6244. The overwhelming majority of phosphosites were responsive to both inhibitors. Of the phosphosites that decreased in abundance, 13 responded only to PLX4032, suggesting that certain targets are regulated by BRAF but not MKK1/2. In contrast, we failed to identify any phosphosites that responded only to AZD6244. To determine whether the therapeutic benefit of combined BRAF and MKK1/2 inhibition may be a result of drug synergy, we treated cells either with a single inhibitor or with a combination of AZD6244 and PLX4032, each at submaximal doses (30 nM). We then compared the magnitude of the responses observed with the combination treatment to those observed for each drug alone. Comparison of observed versus predicted changes at 6,463 phosphosites demonstrated that the primary effect of the two inhibitors is additive. Taken together, we show that the combination of BRAF and MKK1/2 inhibitor leads to predominantly additive interactions, and that the BRAF inhibitor has a potential for regulating phosphorylation events that are not targeted by MKK1/2 inhibitor. Citation Format: Scott Stuart, Stephane Houel, Nan Wang, William Old, Natalie Ahn. A phosphoproteomic comparison of oncogenic BRAF and MKK1/2 inhibition in human melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B24.

Improving Blood Plasma Glycoproteome Coverage by Coupling Ultracentrifugation Fractionation to Electrostatic Repulsion-Hydrophilic Interaction Chromatography Enrichment.

Blood plasma is considered to be an excellent source of disease biomarkers because it contains proteins, lipids, metabolites, cell, and cell-derived extracellular vesicles from different cellular origins including diseased tissues. Most secretory and membranous proteins that can be found in plasma are glycoproteins; therefore, the plasma glycoproteome is one of the major subproteomes that is highly enriched with disease biomarkers. As a result, the glycoproteome has attracted much attention in clinical proteomic research. The modification of proteins with glycans regulates a wide range of functions in biology, but profiling plasma glycoproteins on a global scale has been hampered by the presence of low stoichiometry of glycoproteins in a complex high abundance plasma proteome background and lack of effective analytical technique. This study aims to improve plasma glycoproteome coverage using pig plasma as a model sample with a two-step strategy. The first step involves fractionation of the plasma proteins using ultracentrifugation into supernatant and pellet that is believed to contain low abundant glycoproteins. In the second step, further enrichment of glycopeptides was achieved in both fractions by adopting electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled to tandem mass spectrometry (LC-MS/MS) analysis. The coverage of enriched glycoproteins in supernatant, pellet, and whole plasma sample as control was compared. Using this simple sample fractionation approach by ultracentrifugation and further ERLIC enrichment technique, sample complexity was reduced and glycoproteome coverage was significantly enhanced in supernatant and pellet fractions (by >50%) compared with whole plasma sample. This study showed that when ultracentrifugation is coupled to ERLIC glycopeptides enrichment and glycoproteome identification are significantly improved. This study demonstrates the combination of ultracentrifugation and ERLIC as a useful method for discovering plasma glycoprotein disease biomarkers.

Simultaneous Enrichment of Plasma Soluble and Extracellular Vesicular Glycoproteins Using Prolonged Ultracentrifugation-Electrostatic Repulsion-hydrophilic Interaction Chromatography (PUC-ERLIC) Approach*

Plasma glycoproteins and extracellular vesicles represent excellent sources of disease biomarkers, but laboratory detection of these circulating structures are limited by their relatively low abundance in complex biological fluids. Although intensive research has led to the development of effective methods for the enrichment and isolation of either plasma glycoproteins or extracellular vesicles from clinical materials, at present it is not possible to enrich both structures simultaneously from individual patient sample, a method that affords the identification of biomarker combinations from both entities for the prediction of clinical outcomes will be clinically useful. We have therefore developed an enrichment method for use in mass spectrometry-based proteomic profiling that couples prolonged ultracentrifugation with electrostatic repulsion-hydrophilic interaction chromatography, to facilitate the recovery of both glycoproteins and extracellular vesicles from nondepleted human plasma. Following prolonged ultracentrifugation, plasma glycoproteins and extracellular vesicles were concentrated as a yellow suspension, and simultaneous analyses of low abundant secretory and vesicular glycoproteins was achieved in a single LC-MS/MS run. Using this systematic prolonged ultracentrifugation-electrostatic repulsion-hydrophilic interaction chromatography approach, we identified a total of 127 plasma glycoproteins at a high level of confidence (FDR ≤ 1%), including 48 glycoproteins with concentrations ranging from pg to ng/ml. The novel enrichment method we report should facilitate future human plasma-based proteome and glycoproteome that will identify novel biomarkers, or combinations of secreted and vesicle-derived biomarkers, that can be used to predict clinical outcomes in human patients.

Anion-exchange chromatography of phosphopeptides: weak anion exchange versus strong anion exchange and anion-exchange chromatography versus electrostatic repulsion-hydrophilic interaction chromatography.

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.

Development of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks.

Phosphorylation is an important post-translational modification that is involved in regulating many signaling pathways. Of particular interest are the growth factor mediated Ras and phosphoinositide 3-kinase (PI3K) signaling pathways which, if misregulated, can contribute to the progression of cancer. Phosphoproteomic methods have been developed to study regulation of signaling pathways; however, due to the low stoichiometry of phosphorylation, understanding these pathways is still a challenge. In this study, we have developed a multi-dimensional method incorporating electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with tandem IMAC/TiO2 enrichment for subsequent phosphopeptide identification by LC/MS/MS. We applied this method to PDGF-stimulated NIH 3T3 cells to provide over 11,000 unique phosphopeptide identifications. Upon motif analysis, IMAC was found to enrich for basophilic kinase substrates while the subsequent TiO2 step enriched for acidophilic kinase substrates, suggesting that both enrichment methods are necessary to capture the full complement of kinase substrates. Biological functions that were over-represented at each PDGF stimulation time point, together with the phosphorylation dynamics of several phosphopeptides containing known kinase phosphorylation sites, illustrate the feasibility of this approach in quantitative phosphoproteomic studies.

Multidimensional electrostatic repulsion–hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research

Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research.

Highly sensitive phosphoproteomics by tailoring solid-phase extraction to electrostatic repulsion-hydrophilic interaction chromatography.

In the past decade, several strategies for comprehensive phosphoproteome analysis have been introduced. Most of them combine different phosphopeptide enrichment techniques and require starting material in the milligram range, as a consequence of their insufficient sensitivity. This limitation impairs the applicability of phosphoproteomics to a wide variety of clinical research, where sample material is highly limited. Here we introduce a highly sensitive and easy-to-establish 2D bottom-up strategy for microgram-scale phosphoproteomics, based on electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), a simple solid-phase extraction step by strong cation exchange (SCX) or reversed phase (RP), and LC-MS analysis. With only 100 μg of tryptic digested, nonstimulated HeLa protein and 45 h of LC-MS analysis time, we identified ≥7500 nonredundant and highly confident phosphorylation sites (per replicate). We assigned all phosphorylation sites to 3013 phosphoproteins, covering the entire dynamic range from 10(7) down to a few copies per cell. Compared to affinity-based-enrichment methods using Ti(4+), our ERLIC-based strategy enriched considerably longer and more acidic phosphopeptides and consequently, we identified 327 phosphorylated C-terminal peptides. The simplicity and high sensitivity of ERLIC-SCX/RP-LC-MS render its future promising for microgram-scale-phosphoproteomics in biological, biomedical, and clinical research.

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

Characterization and studies of proteome are challenging because biological samples are complex, with a wide dynamic range of abundance. At present the proteins are identified by digestion into peptides, with subsequent identification of the peptides by mass spectrometry (MS). MS is a powerful technique for the purpose, but it cannot identify every peptide in such complex mixtures simultaneously. For accurate analysis and quantification it is important to separate the peptides first by chro- matography into fractions of a size that MS can handle. With these less complex fractions, the probability is increased of identi- fying peptides of low abundance that would otherwise experience ion suppression effects due to the presence of peptides of high abundance. Enrichment for peptides with certain post-translational modifications helps to increase their detection rates as well. Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is a mixed-mode chromatographic technique which combines the use of electrostatic repulsion and hydrophilic interaction. This review provides an overview of ERLIC and its var- ious proteomics applications. ERLIC has been demonstrated to have good orthogonality to reverse phase liquid chromatography (RPLC), making it useful as a first dimension in multidimensional liquid chromatography (MDLC) and fractionation of digests in general. Peptides elute in order of their isoelectric points and polarity. ERLIC has also been successfully utilized for the enrichment for phosphopeptides and glycopeptides, facilitating their identification. In addition, it is promising for the study of peptide deamidation. ERLIC performs comparably well or better than established methods for these various applications, and serves as a viable and efficient workflow alternative.

Hypoxia-induced Changes to Integrin α 3 Glycosylation Facilitate Invasion in Epidermoid Carcinoma Cell Line A431

Hypoxia is a critical microenvironmental factor that drives cancer progression through angiogenesis and metastasis. Glycoproteins, especially those on the plasma membrane, orchestrate this process; however, questions remain regarding hypoxia-perturbed protein glycosylation in cancer cells. We focused on the effects of hypoxia on the integrin family of glycoproteins, which are central to the cellular processes of attachment and migration and have been linked with cancer in humans. We employed electrostatic repulsion hydrophilic interaction chromatography coupled with iTRAQ labeling and LC-MS/MS to identify and quantify glycoproteins expressed in A431. The results revealed that independent of the protein-level change, N-glycosylation modifications of integrin α 3 (ITGA3) were inhibited by hypoxia, unlike in other integrin subunits. A combination of Western blot, flow cytometry, and cell staining assays showed that hypoxia-induced alterations to the glycosylation of ITGA3 prevented its efficient translocation to the plasma membrane. Mutagenesis studies demonstrated that simultaneous mutation of glycosites 6 and 7 of ITGA3 prevented its accumulation at the K562 cell surface, which blocked integrin α 3 and β 1 heterodimer formation and thus abolished ITGA3's interaction with extracellular ligands. By generating A431 cells stably expressing ITGA3 mutated at glycosites 6 and 7, we showed that lower levels of ITGA3 on the cell surface, as induced by hypoxia, conferred an increased invasive ability to cancer cells in vitro under hypoxic conditions. Taken together, these results revealed that ITGA3 translocation to the plasma membrane suppressed by hypoxia through inhibition of glycosylation facilitated cell invasion in A431.