Separation Of Peptides Research Articles

Daylily protein constituents of the pollen and stigma a proteomics approach

This study was aimed at the identification and quantification of the protein components of the pollen grains in parallel with the distal stigmatic tissue of tetraploid cultivars. Proteomes were analyzed using iTRAQ 4plex labeling, peptides separation by online RP-nano-LC and analysis by ESI–MS/MS. Protein identification and quantification were made using the Asparagales database as a reference. A total of 524,037 MS/MS spectra were produced from pollen and stigma samples. From these, a total of 8368 peptides wereidentified corresponding to 994 unique peptides and 432 protein groups. Among them, 128 differentially expressed proteins were retained for further analysis. In absence of the daylily genome availability, we exploited numerous databases and bioinformatics resources to exploring the putative biological functions of these proteins. The profile of differentially expressed proteins suggests an important representation of functions associated to the signalling and response against endogenous and environmental stresses, including several enzymes implicated in the biosynthesis of antibiotics. The abundance in stigma of several structural proteins of the ribosomal sub-units as well as of the core histones suggest that the translation processes and the regulation of gene expression in stigma is a more active mechanism than in pollen. In addition, pollen prioritizes the synthesis of fructose and glucose as opposed to sucrose in stigma as a source of energy. Finally, the modulated proteins in Hemerocallis point to several pathways that give potential clues concerning the molecular mechanisms underlying the functions of the pollen and the stigmatic fluid in daylily reproduction.

New Baseline Correction Method Using Near-Infrared Absorption of Water in Water/Acetonitrile Gradient High-Performance Liquid Chromatography with Far-Ultraviolet Absorbance Detection

We have previously reported the development of a far-ultraviolet (FUV) absorbance detector capable of detecting wavelengths down to 175 nm in high-performance liquid chromatography (HPLC). Although the FUV detector can detect substances with weak to non-existent ultraviolet (UV) absorption (e.g., sugars at 185 nm and peptides at 190 nm), a large baseline drift occurs in the gradient elution due to differences in the FUV absorbance properties of water and acetonitrile. To overcome the problem of baseline drift, we have proposed a new baseline correction method using the absorption of water at 1450 nm. It is well known that water has a relatively large absorption peak at 1450 nm in the near-infrared (NIR) region. By contrast, acetonitrile used in reversed-phase HPLC shows negligible absorbance compared to water at 1450 nm. Sugars and peptides also show negligible absorbance at 1450 nm. Thus, it is expected that changes in absorbance at 1450 nm only reflect the volume fraction of water in the gradient elution. The baseline correction method by a linear combination of FUV and NIR chromatograms was applied to the HPLC separation of sugars and peptides in water/acetonitrile-gradient HPLC coupled with FUV detection. The results showed that flat baselines were successfully obtained in the gradient HPLC coupled with FUV detection.

Predicting Electrophoretic Mobility of Tryptic Peptides for High-Throughput CZE-MS Analysis

A multiparametric sequence-specific model for predicting peptide electrophoretic mobility has been developed using large-scale bottom-up proteomic CE-MS data (5% (∼0.8M) acetic acid as background electrolyte). Peptide charge (Z) and size (molecular mass, M) are the two major factors determining electrophoretic mobility, in complete agreement with previous studies. The extended size of the data set (>4000 peptides) permits access to many sequence-specific factors that impact peptide mobility. The presence of acidic residues Asp and Glu near the peptide N-terminus is by far the most prominent among them. The induction effect of the side chain of N-terminal Asp reduces the basicity of the N-terminal amino group and, as hence, its charge, by ∼0.27 units, lowering mobility. The correlation of the model (R2 ∼ 0.995) indicates that the peptide separation process in CZE is relatively simple and can be predicted to a much higher precision than current RP-HPLC models. Similar to RP-HPLC prediction studies, we anticipate future developments that introduce peptide migration standards, collect larger data sets for modeling through the alignment of multiple CZE-MS acquisitions, and study of the behavior of peptides carrying post-translational modifications. The increased size of data sets will also permit investigation of the fine-scale effects of peptide secondary structure on peptide mobility. We observed that peptides with higher helical propensity tend to have higher than predicted electrophoretic mobility; the incorporation of these features into CZE migration models will require significantly larger data sets.

High-Speed, Comprehensive, Two Dimensional Separations of Peptides and Small Molecule Biological Amines Using Capillary Electrophoresis Coupled with Micro Free Flow Electrophoresis.

Two-dimensional (2D) separations are able to generate significantly higher peak capacities than their one-dimensional counterparts. Unfortunately, current hyphenated 2D separations are limited by the speed of the second dimension separation and the consequent loss of peak capacity due to under sampling of peaks as they elute from the first dimension separation. Continuous micro free flow electrophoresis (μFFE) separations eliminate under sampling as a limitation when incorporated as the second dimension of a 2D separation. In the current manuscript we describe the first coupling of capillary electrophoresis (CE) with μFFE to perform 2D CE × μFFE separations. The CE separation capillary was directly inserted into the μFFE separation channel using an edge on interface. Analyte peaks streamed directly into the μFFE separation channel as they migrated off the CE capillary. No complicated injection, valving, or voltage changes were necessary to couple the two separation modes. 2D CE × μFFE generated an ideal peak capacity of 2 592 in a 9 min separation of fluorescently labeled peptides (7.6 min separation window, 342 peaks/min). Data points were recorded every 250-500 ms (>8 data points/peak), effectively eliminating under sampling as a source of band broadening. CE × μFFE generated an ideal peak capacity of 1885 in a 2.7 min separation of fluorescently labeled small molecule bioamines (1.8 min separation window, 1053 peaks/min). Peaks in the 2D CE × μFFE separation of peptides covered 30% of the available separation space, resulting in a corrected peak capacity of 778 (102 peaks/min). The fractional coverage of the 2D CE × μFFE separation of small molecule bioamines was 20%, resulting in a corrected peak capacity of 377 (209 peaks/min).

The Separation and Quantitation of Peptides with and without Oxidation of Methionine and Deamidation of Asparagine Using Hydrophilic Interaction Liquid Chromatography with Mass Spectrometry (HILIC-MS).

Peptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein's function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts. Furthermore, coefficients describing the extent of the hydrophilicity of these modifications have been derived and were incorporated into a previously made peptide retention prediction model that is capable of predicting the retention times of peptides with and without these modifications. Graphical Abstract ᅟ.

High pH Reversed-Phase Micro-Columns for Simple, Sensitive, and Efficient Fractionation of Proteome and (TMT labeled) Phosphoproteome Digests.

Despite recent advances in mass spectrometric sequencing speed and improved sensitivity, the in-depth analysis of proteomes still widely relies on off-line peptide separation and fractionation to deal with the enormous molecular complexity of shotgun digested proteomes. While a multitude of methods has been established for off-line peptide separation using HPLC columns, their use can be limited particularly when sample quantities are scarce. In this protocol, we describe an approach which combines high pH reversed-phase peptide separation into few fractions in StageTip micro-columns. This miniaturized sample preparation method enhances peptide recovery and hence improves sensitivity. This is particularly useful when working with limited sample amounts obtained from e.g., phosphopeptide enrichments or tissue biopsies. Essentially the same approach can also be applied for multiplexed analysis using tandem mass tags (TMT) and can be parallelized in order to deliver the required throughput. Here, we provide a step-by-step protocol for TMT6plex labeling of peptides, the construction of StageTips, sample fractionation and pooling schemes adjusted to different types of analytes, mass spectrometric sample measurement, and downstream data processing using MaxQuant. To illustrate the expected results using this protocol, we provide results from an unlabeled and a TMT6plex labeled phosphopeptide sample leading to the identification of >17,000 phosphopeptides in 8 h (Q Exactive HF) and >23,000 TMT6plex labeled phosphopeptides (Q Exactive Plus) in 12 h of measurement time. Importantly, this protocol is equally applicable to the fractionation of full proteome digests.

Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation.

Studying liquid-liquid phase separation (LLPS) of proteins provides key insights into the biogenesis of membraneless organelles and pathological protein aggregation in disease. We have established a protocol for inducing the phase separation of arginine-rich peptides, which allows for studying their molecular determinants and dynamics ( Boeynaems et al., 2017 ).

Hydrophilic Strong Anion Exchange (hSAX) Chromatography Enables Deep Fractionation of Tissue Proteomes.

The bottom-up proteomic analysis of cell line and tissue samples to a depth > 10,000 proteins still represents a considerable challenge because of the sheer number of peptides generated by proteolytic digestions and the high dynamic range of protein expression. As a result, comprehensive protein coverage requires multidimensional peptide separation. Recently, off-line hydrophilic strong cation exchange (hSAX) chromatography has proven its merits for high resolution separation of peptides due to its high degree of orthogonality to reversed-phase liquid chromatography. Here we describe the use of hSAX for the deep analysis of tissue proteomes. The protocol includes optimized sample preparation steps (lysis with the aid of mechanical disruption, one-step disulfide bridge reduction and alkylation), setup and operation of hSAX columns and gradients, desalting of hSAX fractions prior to LC-MS/MS analysis, and suggestions for the choice of data acquisition parameters and data analysis using MaxQuant. Application of the protocol to the fractionation of 300 μg human brain tissue digest led to the identification of more than 100,000 unique peptide sequences representing over 10,195 proteins and 9,500 genes in 3 days of measurement time on a Q Exactive Plus mass spectrometer.

Plant Cell Wall Proteomics: An Assessment Twenty Years after Launching

Cell walls are complex structures surrounding plant cells. They provide not only mechanical support and protection against environmental changes, but also a mean for cell-to-cell communication. They are mainly constituted of polysaccharides (about 90% of their mass) and proteins. Cell wall proteins (CWPs) play critical roles because they contribute to the plasticity of the cell wall architecture during development and in response to biotic and abiotic environmental changes. Their systematic identification has started in the 2000’s with the sequencing of the genome of the Arabidopsis thaliana model plant and the development of adapted mass spectrometry (MS) technologies. Since then, many other plants have been studied among which plants of agronomical interest. The description of cell wall proteomes has fully benefited not only from the improvement of MS technologies, but also from better sample preparation and peptide separation prior to MS analysis. Bioinformatics has also played critical roles by designing software allowing protein identification, annotation and quantification, as well creating MS data repositories.

Influence of NanoLC Column and Gradient Length as well as MS/MS Frequency and Sample Complexity on Shotgun Protein Identification of Marine Bacteria

Protein identification by shotgun proteomics, i.e., nano-liquid chromatography (nanoLC) peptide separation online coupled to electrospray ionization (ESI) mass spectrometry (MS)/MS, is the most widely used gel-free approach in proteome research. While the mass spectrometer accounts for mass accuracy and MS/MS frequency, the nanoLC setup and gradient time influence the number of peptides available for MS analysis, which ultimately determine the number of proteins identifiable. Here, we report on the influence of (i) analytical column length (15, 25, or 50 cm) coupled to (ii) the applied gradient length (120, 240, 360, 480, or 600 min), as well as (iii) MS/MS frequency on peptide/protein identification by shotgun proteomics of (iv) 2 marine bacteria. Longer gradients increased the number of peptides/proteins identified as well as the reproducibility of identification. Furthermore, longer analytical columns strictly enlarge the covered proteome complement. Notably, the proteome complement identified with a short column and applying a long gradient is also covered when using longer columns with shorter gradients. Coverage of the proteome complement further increases with higher MS/MS frequency. Compilation of peptide lists of replicate analyses (same gradient length) improves protein identification, while compilation of analyses with different gradient lengths yields a similar or even higher number of proteins using comparable or even less total analysis time.

Discovery and Longitudinal Evaluation of Candidate Biomarkers for Ischaemic Stroke by Mass Spectrometry-Based Proteomics.

Application of acute therapies such as thrombolysis for ischaemic stroke (IS) is constrained because of diagnostic uncertainty and the dynamic nature of stroke biology. To investigate changes in blood proteins after stroke and as a result of thrombolysis treatment we performed label-free quantitative proteomics on serum samples using high-resolution mass spectrometry and long high-performance liquid chromatography gradient (5 hours) combined with a 50-cm column to optimise the peptide separation. We identified (false discovery rate [FDR]: 1%) and quantified a total of 574 protein groups from a total of 92 samples from 30 patients. Ten patients were treated by thrombolysis as part of a randomised placebo-controlled trial and up to 5 samples were collected from each individual at different time points after stroke. We identified 26 proteins differently expressed by treatment group (FDR: 5%) and significant changes of expression over time for 23 proteins (FDR: 10%). Molecules such as fibrinogen and C-reactive protein showed expression profiles with a high-potential clinical utility in the acute stroke setting. Protein expression profiles vary acutely in the blood after stroke and have the potential to allow the construction of a stroke clock and to have an impact on IS treatment decision making.

Contribution of Eigenmobility Shifts to the Separation of Peptides in Capillary Electrophoresis with Aqueous–Acetonitrile Background Electrolytes

In this investigation, the mobility of system eigenpeaks in capillary electrophoresis (CE) was experimentally found to decrease when the background electrolyte (BGE) contained higher percentages of acetonitrile. In order to explain this observation, the effects of changes in the pH and ionic strength of the BGE on the pKa and actual mobility of each constituent in the system were determined, and the results evaluated in terms of their theoretical basis. Utilizing the derived values of each of these parameters, the software Peakmaster was then applied to simulate the eigenpeak mobility. Although general trends for BGEs with different acetonitrile contents could be simulated, these simulations did not exactly match the experimental results. To account for this divergence between theory and experimental practice, the consequences of tube radial distribution of the organic solvent in an aqueous-organic system within the capillary and the effects of radial ion distribution leading to the electro-osmotic flow mobility (EOF) are proposed to be the cause of this deviation. Consequently, the Debye-Hückel approximation and Boltzmann distribution function were employed to calculate the amount of each constituent across the radius of the capillary. The inhomogeneous radial distributions of the constituents in the BGE and the organic solvent were simplified to a 1-dimensional problem based on a 4-constituent BGE approximation. A high level of correlation was then achieved between the experimental results and the corresponding CE separations simulated using Peakmaster. In addition, cancellation or suppression of the peak broadening was experimentally and theoretically demonstrated by taking advantage of the influence of a second independent system eigenpeak. The outcome from these studies was a new way to achieve sharpening of specific peaks in the CE separations of peptides.

Applicability of the critical chromatography concept to proteomic problems. II. Effect of mobile phase on the separation of peptides and proteins taking into account the amino acid sequence

The effect of parameters of a binary mobile phase (chemical properties of components, gradient profile, flow rate, and pH) on the separation of peptides and proteins is theoretically investigated using a model of critical chromatography (model BioLCCC). It is demonstrated that the retention volume of peptides can be inverted by changing separation conditions.

Label-free quantitative comparison of cerebrospinal fluid glycoproteins and endogenous peptides in subjects with Alzheimer's disease, mild cognitive impairment, and healthy individuals.

The goal of this study is to investigate putative molecular dynamic changes in cerebrospinal fluids (CSFs) collected from individuals with mild cognitive impairment (MCI) and Alzheimer's disease (AD) as compared to healthy controls. The CSF samples from 12 subjects comprised of four cognitively normal individuals and eight patients with MCI and AD, respectively. Two aliquots of each CSF samples (total 1 mL) of each participant are used for this study. Endogenous peptide separations are performed using 10000 molecular weight cut-off filters followed by LC-MS/MS identification and quantitation while lectin-enrichment chromatography is used to enrich glycoproteins in CSF followed by trypsin digestion and subsequent LC-MS/MS for shotgun identification and label-free quantitation. Using an optimized submicrogram peptide separation with molecular weight cut-off filtration and an in house-constructed database, 645 peptides are identified. Glycoproteins are enriched by lectin affinity chromatography, resulting in 795 identified proteins. The discovery and alterations of proSAAS-derived peptides and transthyretin are described and their roles in AD are discussed. Comprehensive identification of endogenous CSF peptidome is achieved. Fifteen proteins are found to be differentially expressed among the three groups. The dynamic changes of transthyretin are reported for the first time.

An enlarged cell wall proteome of Arabidopsis thaliana rosettes.

Plant cells are surrounded by cell walls playing many roles during development and in response to environmental constraints. Cell walls are mainly composed of polysaccharides (cellulose, hemicelluloses and pectins), but they also contain proteins which are critical players in cell wall remodeling processes. Today, the cell wall proteome of Arabidopsis thaliana, a major dicot model plant, comprises more than 700 proteins predicted to be secreted (cell wall proteins-CWPs) identified in different organs or in cell suspension cultures. However, the cell wall proteome of rosettes is poorly represented with only 148 CWPs identified after extraction by vacuum infiltration. This new study allows enlarging its coverage. A destructive method starting with the purification of cell walls has been performed and two experiments have been compared. They differ by the presence/absence of protein separation by a short 1D-electrophoresis run prior to tryptic digestion and different gradient programs for peptide separation before mass spectrometry analysis. Altogether, the rosette cell wall proteome has been significantly enlarged to 361 CWPs, among which 213 newly identified in rosettes and 57 newly described. The identified CWPs fall in four major functional classes: 26.1% proteins acting on polysaccharides, 11.1% oxido-reductases, 14.7% proteases and 11.7% proteins possibly related to lipid metabolism.

Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain.

The fusion peptide (FP) and transmembrane domain (TMD) of viral fusion proteins play important roles during virus-cell membrane fusion, by inducing membrane curvature and transient dehydration. The structure of the water-soluble ectodomain of viral fusion proteins has been extensively studied crystallographically, but the structures of the FP and TMD bound to phospholipid membranes are not well understood. We recently investigated the conformations and lipid interactions of the separate FP and TMD peptides of parainfluenza virus 5 (PIV5) fusion protein F using solid-state nuclear magnetic resonance. These studies provide structural information about the two domains when they are spatially well separated in the fusion process. To investigate how these two domains are structured relative to each other in the postfusion state, when the ectodomain forms a six-helix bundle that is thought to force the FP and TMD together in the membrane, we have now expressed and purified a chimera of the FP and TMD, connected by a Gly-Lys linker, and measured the chemical shifts and interdomain contacts of the protein in several lipid membranes. The FP-TMD chimera exhibits α-helical chemical shifts in all the membranes examined and does not cause strong curvature of lamellar membranes or membranes with negative spontaneous curvature. These properties differ qualitatively from those of the separate peptides, indicating that the FP and TMD interact with each other in the lipid membrane. However, no 13C-13C cross peaks are observed in two-dimensional correlation spectra, suggesting that the two helices are not tightly associated. These results suggest that the ectodomain six-helix bundle does not propagate into the membrane to the two hydrophobic termini. However, the loosely associated FP and TMD helices are found to generate significant negative Gaussian curvature to membranes that possess spontaneous positive curvature, consistent with the notion that the FP-TMD assembly may facilitate the transition of the membrane from hemifusion intermediates to the fusion pore.

Capillary methacrylate-based monoliths by grafting from/to γ-ray polymerization on a tentacle-type reactive surface for the liquid chromatographic separations of small molecules and intact proteins

Capillary methacrylate-based monoliths were prepared for the high performance liquid chromatography (HPLC) separation of both small molecules and large biomolecules. An efficient grafting from/to synthetic approach was adopted introducing a network of activated sites in the inner wall surface using the new silanization agent (N-trimethoxysilylpropyl)-polyethylenimine. Copolymerization of lauryl methacrylate monomer and 1,6-hexanediol dimethacrylate cross-linker in the presence of porogenic solvents was obtained under continuous γ-ray exposure with high conversion yield.The morphology and porous structure of the resulting monoliths have been investigated by Scanning Electron Microscopy (SEM) and 1H NMR cryoporosimetry. By chromatographic investigation, the new capillary columns attested high kinetic performance (with efficiency larger than 100,000 theoretical plate/m for small molecules at optimum mobile phase linear velocity of about 0.5mm/s) and also excellent mechanical stability and repeatability.The new methacrylate-based monolithic capillary columns have been successfully employed for efficient reversed-phase separation of intact proteins and peptides.

Generation of accurate peptide retention data for targeted and data independent quantitative LC-MS analysis: Chromatographic lessons in proteomics.

The emergence of data-independent quantitative LC-MS/MS analysis protocols further highlights the importance of high-quality reproducible chromatographic procedures. Knowing, controlling and being able to predict the effect of multiple factors that alter peptide RP-HPLC separation selectivity is critical for successful data collection for the construction of ion libraries. Proteomic researchers have often regarded RP-HPLC as a "black box", while vast amount of research on peptide separation is readily available. In addition to obvious parameters, such as the type of ion-pairing modifier, stationary phase and column temperature, we describe the "mysterious" effects of gradient slope, column size and flow rate on peptide separation selectivity. Retention time variations due to these parameters are governed by the linear solvent strength (LSS) theory on a peptide level by the value of its slope S in the basic LSS equation-a parameter that can be accurately predicted. Thus, the application of shallower gradients, higher flow rates, or smaller columns will each increases the relative retention of peptides with higher S-values (long species with multiple positively charged groups). Simultaneous changes to these parameters that each drive shifts in separation selectivity in the same direction should be avoided. The unification of terminology represents another pressing issue in this field of applied proteomics that should be addressed to facilitate further progress.

Separation of Peptides with Forward Osmosis Biomimetic Membranes.

Forward osmosis (FO) membranes have gained interest in several disciplines for the rejection and concentration of various molecules. One application area for FO membranes that is becoming increasingly popular is the use of the membranes to concentrate or dilute high value compound solutions such as pharmaceuticals. It is crucial in such settings to control the transport over the membrane to avoid losses of valuable compounds, but little is known about the rejection and transport mechanisms of larger biomolecules with often flexible conformations. In this study, transport of two chemically similar peptides with molecular weight (Mw) of 375 and 692 Da across a thin film composite Aquaporin Inside™ Membrane (AIM) FO membrane was investigated. Despite the relative large size, both peptides were able to permeate the dense active layer of the AIM membrane and the transport mechanism was determined to be diffusion-based. Interestingly, the membrane permeability increased 3.65 times for the 692 Da peptide (1.39 × 10−12 m2·s−1) compared to the 375 Da peptide (0.38 × 10−12 m2·s−1). This increase thus occurs for an 85% increase in Mw but only for a 34% increase in peptide radius of gyration (Rg) as determined from molecular dynamics (MD) simulations. This suggests that Rg is a strong influencing factor for membrane permeability. Thus, an increased Rg reflects the larger peptide chains ability to sample a larger conformational space when interacting with the nanostructured active layer increasing the likelihood for permeation.

Analysis of proteins and peptides by electromigration methods in microchips

This review presents the developments and applications of microchip electromigration methods in the separation and analysis of peptides and proteins in the period 2011-mid-2016. The developments in sample preparation and preconcentration, microchannel material, and surface treatment are described. Separations by various microchip electromigration methods (zone electrophoresis in free and sieving media, affinity electrophoresis, isotachophoresis, isoelectric focusing, electrokinetic chromatography, and electrochromatography) are demonstrated. Advances in detection methods are reported and novel applications in the areas of proteomics and peptidomics, quality control of peptide and protein pharmaceuticals, analysis of proteins and peptides in biomatrices, and determination of physicochemical parameters are shown.