Separation Of Complex Peptide Mixtures Research Articles

Benzyl isothiocyanate-modified α-lactalbumin – Two-dimensional high-performance thin-layer chromatography for analyzing modified peptides

In complex food matrices, non-directed reactions between food proteins and secondary plant metabolites (SPM) are conceivable. In this study, the interaction between the bioactive metabolite from garden cress (Lepidium sativum) and selected Brassicaceae – benzyl isothiocyanate (BITC) – and the dairy protein α-lactalbumin (α-LA) was investigated. It was focused on monitoring the proteolytic degradation behaviour of unmodified and BITC-modified α-LA with two-dimensional high-performance thin-layer chromatography (2D-HPTLC). The two-dimensional approach of HPTLC offers high resolution in the separation of complex peptide mixtures and might enable differentiation of protein modifications. Based on the specific peptide patterns of native and modified peptides, conclusions can be drawn about differences in protein/peptide polarity, location of a modification, and digestibility. The aim was to characterize tryptically hydrolyzed unmodified and BITC-modified peptides using the 2D method and to investigate the influence of BITC modification of α-LA on polarity and digestibility. To determine the repeatability of peptide separation by 2D-HPTLC, the unmodified and BITC-modified protein hydrolyzates were separated six times. The absolute standard deviations between the retardation factors of the individual peptide spots varied between 0.52 and 4.79 mm for the x-coordinates and between 0.41 and 6.47 mm for the y-coordinates for all three samples. Here, the mean relative standard deviations ranged from 5.80 to 10.4% for the x-coordinates and from 5.91 to 18.3% for the y-coordinates. The results of the tryptic hydrolysis indicated that, depending on the concentration of BITC used, the modification sterically hinders the cleavage sites for the enzyme, resulting in a reduced digestibility. Covalent binding of the hydrophobic BITC altered the digestibility and polarity of the protein, leading to a difference in peptide patterns between the unmodified and modified α-LA. It was concluded that the reaction was undirected, resulting in a mixture of unmodified and modified peptides, and that elongated modified peptides were formed by BITC blocking of trypsin cleavage sites.

Two-dimensional high-performance thin-layer chromatography for the characterization of milk peptide properties and a prediction of the retention behavior – a proof-of-principle study

High-performance thin-layer chromatography (HPTLC) is a suitable method for the analysis of peptides and proteins due to a wide selection of stationary and mobile phases and various detection options. Especially, two-dimensional HPTLC (2D-HPTLC) enables a higher resolution compared to one-dimensional HPTLC in the separation of complex peptide mixtures. Similar to 2D electrophoresis, characteristic peptide patterns can be obtained, allowing a differentiation of ingredients based on varying protein origins.The aim of this study was to evaluate 2D-HPTLC with regard to its suitability for the characterization of proteins/peptides and to verify whether it is possible to predict the retention behavior of peptides based on their properties. As models, the five most abundant milk proteins α-lactalbumin, β-lactoglobulin, α-, β-, and κ-Casein were used. In order to determine the repeatability of the peptide separation by 2D-HPTLC, each tryptic protein hydrolyzate was separated eight times. The standard deviations of the retardation factors for the separated peptides varied between 1.0 and 11.1 mm for the x-coordinate and 0.5–7.3 mm for the y-coordinate. It was also shown that after the chromatographic separation, peptides of the individual protein hydrolyzates were located in specific areas on the HPTLC plate, so that a clustering could be obtained for the whey proteins‘ as well as the caseins‘ hydrolyzates. For establishing correlations between the properties of the peptides and their retardation factors, 51 of 85 selected peptides were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). On this basis, statistically significant correlations (α = 0.05) between the retardation factors of the peptides and their isoelectric points, as well as the percentage of anionic and non-polar amino acids in the peptides were established. Finally, it was investigated, whether the retardation factors for peptides can be predicted on the basis of a linear regression of the percentage of non-polar amino acids in a peptide. For this purpose, a mixture of artifical (synthetic) peptides (n = 14) was separated by 2D-HPTLC and the measured retardation factors were compared with the corresponding retardation factors calculated. Absolute deviations of 0.3–17.9 mm were obtained. In addition, the universal applicability of the method to other protein sources other than milk proteins (animal protein) was tested using a mixture of pea peptides (plant protein, n = 3) resulting in absolute deviations of 0.7–8.6 mm.

Predictive Liquid Chromatography of Peptides Based on Hydrophilic Interactions for Mass Spectrometry-Based Proteomics

High-performance liquid chromatography (HPLC) is widely used for separation of complex peptide mixtures before mass spectrometry-based proteome analysis. In this analysis, reversed phase HPLC (RPHPLC) using non-polar stationary phases such as surface-modified silica containing alkyl groups (e.g., C18) is typically employed. Because of the high heterogeneity of proteomic samples, multidimensional separation approaches gained increasing attention recently to tackle this complexity and extremely high range of concentrations. In two-dimensional liquid chromatography, hydrophilic interaction chromatography (HILIC) is often a method of choice for combination with RP-HPLC because it uses reversed-phase type eluents and allows efficient separation of polar peptides. Due to the high degree of orthogonality in this two-dimensional separation space, it is tempting to develop approaches for predicting peptide retention times for HILIC-based separations similar to the ones for RP-HPLC. Recent successful efforts in this area were focused on developing retention coefficient (RC)-based approaches. Herein, we explored the feasibility of using a statistical thermodynamic model for prediction of peptide retention times in HILIC separations and determined the phenomenological parameters of the model for a bare silica column. The performance of the developed model was tested using HPLC-MS analysis of a set of synthetic peptides, as well as a tryptic peptide mixture.

Limitation of predictive 2‐D liquid chromatography in reducing the database search space in shotgun proteomics: In silico studies

A two-dimensional (2-D) liquid chromatography (LC) separation of complex peptide mixtures that combines a normal phase utilizing hydrophilic interactions and a reversed phase offers reportedly the highest level of 2-D LC orthogonality by providing an even spread of peptides across multiple LC fractions. Matching experimental peptide retention times to those predicted by empirical models describing chromatographic separation in each LC dimension leads to a significant reduction in a database search space. In this work, we calculated the retention times of tryptic peptides separated in the C18 reversed phase at different separation conditions (pH 2 and pH 10) and in TSK gel Amide-80 normal phase. We show that retention times calculated for different 2-D LC separation schemes utilizing these phases start to correlate once the mass range of peptides under analysis becomes progressively narrow. This effect is explained by high degree of correlation between retention coefficients in the considered phases.

Open‐tubular capillary electrochromatography with bare gold nanoparticles‐based stationary phase applied to separation of trypsin digested native and glycated proteins

In this work, open-tubular capillary electrochromatography (OT-CEC) method with bare gold nanoparticles (GNPs)-based stationary phase has been developed and applied for separation of tryptic peptide fragments of native and glycated proteins, bovine serum albumin (BSA), and human transferrin (HTF). The GNPs-based stationary phase was prepared by immobilization of bare GNPs, freshly reduced from tetrachloroaurate(III) ions by citrate reduction, on the sol-gel pretreated inner wall of the fused silica capillary. The separation efficiency, peak capacity, and peptide recovery of this open-tubular capillary column were investigated by varying the experimental parameters such as type and concentration of the buffering constituent and pH of the background electrolyte (BGE), temperature, and separation voltage. The best separations of the above tryptic peptides were achieved in the BGE composed of aqueous 100 mmol/L sodium phosphate buffer, pH 2.5, at separation voltage 10 kV per 47-cm long, 50 μm inside diameter capillary thermostated at 25°C. OT-CEC with bare GNPs stationary phase is shown to be a suitable technique for separation of complex peptide mixtures arising from tryptic digestion of native and glycated BSA and HTF, and for investigation of glycation (nonenzymatic glycosylation) of these proteins.

Poly(glycidyl methacrylate-divinylbenzene) based immobilized pH gradient capillary isoelectric focusing coupling with MALDI mass spectrometry for enhanced neuropeptide analysis.

Herein, we report an immobilized pH gradient (IPG) capillary isoelectric focusing-matrix-assisted laser desorption/ionization mass spectrometry (CIEF-MALDI MS) platform designed for the separation of complex neuropeptides. This platform features a poly(glycidyl methacrylate-divinylbenzene) (GMA-DVB)-based monolithic column for CIEF separation. Different from regular CIEF, carrier ampholytes are preimmobilized on the monolithic surface instead of being added to the sample. An off-line coupling of IPG-CIEF to MALDI MS has been established. Comparison with regular CIEF and optimizations are performed with bovine serum albumin tryptic peptides and extracted neuropeptide mixtures from crustacean Callinectes sapidus. It has been demonstrated that the separation of complex peptide mixtures in neutral and basic pH ranges can be achieved in less than 10 min with comparable separation efficiency with regular CIEF, while the MS signal is significantly enhanced when employing IPG-CIEF. Enhanced neuropeptide detection is also observed after coupling IPG-CIEF with MALDI MS.

Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC and ZIC-cHILIC) Provide High Resolution Separation and Increase Sensitivity in Proteome Analysis

The complexity of peptide mixtures that are analyzed in proteomics necessitates fractionation by multidimensional separation approaches prior to mass spectrometric analysis. In this work, we introduce and evaluate hydrophilic interaction liquid chromatography (HILIC) based strategies for the separation of complex peptide mixtures. The two zwitterionic HILIC materials (ZIC-HILIC and ZIC-cHILIC) chosen for this work differ in the spatial orientation of the positive and negative charged groups. Online experiments revealed a pH-independent resolving power for the ZIC-cHILIC resin while ZIC-HILIC showed a decrease in resolving power at an acidic pH. Subsequently, we extensively evaluated the performances of ZIC-HILIC and ZIC-cHILIC as first dimension in an off-line two-dimensional liquid chromatography (2D-LC) strategy in combination with reversed phase (RP), with respect to peptide separation efficiency and how the retention time correlates with a number of peptide physicochemical properties. Both resins allowed the identification of more than 20,000 unique peptides corresponding to over 3500 proteins in each experimental condition from a remarkably low (1.5 μg) amount of starting material of HeLa lysate digestion. The resulting data allows the drawing of a comprehensive picture regarding ZIC- and ZIC-cHILIC peptide separation characteristics. Furthermore, the extent of protein identifications observed from such a level of material demonstrates that HILIC can rival or surpass traditional multidimensional strategies employed in proteomics.

Wider Pore Superficially Porous Particles for Peptide Separations by HPLC

Fused-core superficially porous particles have recently created considerable interest for high-performance liquid chromatography separations because of their unusual high column efficiency and much lower back pressure when compared to sub-2-microm particles. With superficially porous particles, larger solutes can move rapidly in and out of a thin porous shell, resulting in reduced band broadening at higher mobile phase velocities for greater separation speeds. The original silica fused-core particles were 2.7 microm in diameter with a 0.5-microm thick shell of 90 A pores designed for the fast separation of small molecules with molecular weights of less than approximately 5000. This manuscript describes new fused-core particles with similar physical characteristics except with a porous shell of 160 A pores designed specifically for rapidly separating peptides (and some small proteins) with molecular weights up to approximately 15,000 Daltons. Because of the larger pore size, restricted diffusion of these larger molecules is not seen since ready access to the entire porous shell is featured. Data are given to define sample loading qualities for columns of these new particles. Column stability studies indicate that these particles bonded with a sterically protected C(18) stationary phase can be used at low pH and higher temperatures with excellent results. The wider-pore particles of this study are shown to be particularly useful with a mass spectrometer detector for the rapid gradient separation of peptides using both volatile trifluoroacetic acid and formic acid containing mobile phases. Examples are provided for the separation of complex peptide mixtures to illustrate the capabilities for columns of these new wider-pore, fused-core particles.

A 2D reversed-phase × ion-pair reversed-phase HPLC-MALDI TOF/TOF-MS approach for shotgun proteome analysis

The separation of complex peptide mixtures in shotgun proteome analysis using a 2D separation scheme encompassing reversed-phase x ion-pair reversed-phase (IP-RP) liquid chromatography coupled online to electrospray ion trap mass spectrometry (MS) has been shown earlier to be superior in terms of separation efficiency and technical robustness compared to the classically used separation scheme encompassing strong cation exchange x IP-RP-chromatography in shotgun proteome analysis. In the present study, this novel separation scheme was coupled offline to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF-MS for the analysis of the same sample, a tryptic digest of the cytosolic proteome of the bacterium Corynebacterium glutamicum. Compared to the earlier study, the MALDI-based platform led to a significantly increased number of peptides (7,416 vs. 2,709) and proteins (1,208 vs. 468, without single peptide-based identifications), respectively. This represents the majority of all predicted cytosolic proteins in C. glutamicum. The high proteome coverage, as well as the large number of low-abundant proteins identified with this improved analytical platform, pave the way for new biological studies.

CEC separation of peptides using a poly(hexyl acrylate-co-1,4-butanediol diacrylate-co-[2-(acryloyloxy)ethyl]trimethyl ammonium chloride) monolithic column.

A polyacrylate-based monolithic column bearing cationic functionalities and designed for capillary electrochromatography (CEC) has been prepared via photopolymerization of a mixture of hexyl acrylate, butanediol diacrylate, 2-(acryloyloxy) ethyltrimethyl ammonium chloride (monomers), azobisisobutyronitrile (photoinitiator), acetonitrile, phosphate buffer, and ethanol (porogens). The polymerization process was initiated with UV light at 360 nm. The column performance was evaluated via the separations of alkylbenzenes, substituted anilines, basic drugs, peptides, and a protein digest. The separation of complex peptide mixtures was then studied since such separations constitute a promising application of capillary electrochromatography. In particular, the effects of mobile phase composition, including ionic strength of the buffer solution and the percentage of acetonitrile on the retention factor, the column efficiency, and the resolution were determined. The separations were affected by both interaction of the peptides with the stationary phase and their own electrophoretic mobility. Excellent separations with column efficiencies of up to 160 000 plates/m were achieved for both a mixture of ten well-defined peptides and a tryptic digest of cytochrome c. The fractions of eluent containing peptides of the digest separated in the monolithic column were collected and characterized using matrix-assisted laser desorption ionization mass spectrometry.

Targeted Quantitative Analysis of Streptococcus pyogenes Virulence Factors by Multiple Reaction Monitoring

In many studies, particularly in the field of systems biology, it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quantitative approach by which predetermined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quantitative Analysis by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amounts of plasma. The resulting quantitative protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.

Elucidation of N-Glycosylation Sites on Human Platelet Proteins

Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets.

Proteomics of Collagen Peptides: A Method to Reveal Minor Changes in Post‐Translationally Modified Collagen by HPLC and Capillary Electrophoresis

Depository effects in slowly metabolized proteins, typically glycation or the estimation of products arising from the reaction of unsaturated long chain fatty acid metabolites (possessing aldehydic groups) are very difficult to assess owing to their extremely low concentration in the protein matrix. Posttranslational nonenzymatic modifications of collagen with sugars and oxidation products of lipid metabolism in tail tendons of two groups of rats (controls and hypertriglyceridemics with a high fructose diet) were studied. Collagen (a mixture of type I and III) was digested by bacterial collagenase and separated by reversed‐phase HPLC; the profile obtained was divided into five fractions, which were further characterizated by capillary electrophoresis (CE) in a bare fused silica capillary (37/30 cm×75 µm I.D.). As the chromatographic and electromigration behaviour of individual peptides was considerably different, the combination of HPLC and CE appeared as a suitable approach capable of acceptable separation of complex peptide mixtures (over 150 peptide peaks were obtained on the electropherograms). This two‐stepped peptide mapping with subsequent statistical evaluation (linear regression analysis) was shown to represent a reliable approach for revealing posttranslational modifications in slowly metabolized model proteins in vivo.

Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography

Column peak capacity was utilized as a measure of column efficiency for gradient elution conditions. Peak capacity was evaluated experimentally for reversed-phase (RP) and cation-exchange high-performance liquid chromatography (HPLC) columns, and compared to the values predicted from RP-HPLC gradient theory. The model was found to be useful for the prediction of peak capacity and productivity in single- and two-dimensional (2D) chromatography. Both theoretical prediction and experimental data suggest that the number of peaks separated in HPLC reaches an upper limit, despite using highly efficient columns or very shallow gradients. The practical peak capacity value is about several hundred for state-of-the-art RP-HPLC columns. Doubling the column length (efficiency) improves the peak capacity by only 40%, and proportionally increases both the separation time and the backpressure. Similarly, extremely shallow gradients have a positive effect on the peak capacity, but analysis becomes unacceptably long. The model predicts that a 2D-HPLC peak capacity of 15,000 can be achieved in 8 h using multiple fraction collection in the first dimension followed by fast RP-HPLC gradients employing short, but efficient columns in the second dimension.

A miniaturized multichamber solution isoelectric focusing device for separation of protein digests.

A miniaturized multichamber device was constructed for solution isoelectric focusing (IEF) separation of complex peptide mixtures. The system, based on immobilized pH gels, consisted of 96 minichambers ( approximately 75 nuL each) arranged in eight rows. Neighboring chambers in a given row were separated by short glass tubes (4 mm inner diameter, 3 mm long), within which Immobiline gels of specific pH values were polymerized. During focusing, the device was sandwiched between two supporting blocks incorporating the reservoirs for anolyte and catholyte. In principle, multiple samples could be simultaneously fractionated, each separated into 12 fractions of various pI ranges. A variety of standard peptide mixtures and tryptic digests of proteins were separated by IEF using this device, and the fractions were characterized by mass spectrometry. For a codigested nine-protein mixture, both the total number of peptides identified and the average sequence coverage were similar to the results of ion-exchange chromatography (IEC), according to matrix assisted laser/desorption/ionization--time of flight (MALDI-TOF) data. The IEF separation provided concentrated and desalted fractions, suitable for an additional separation liquid chromatography, capillary electrophoresis (LC, CE) or mass spectrometry (MS) detection without additional sample cleanup. High loading capacity was achieved for the miniaturized multichamber IEF device. Importantly, a linear correlation was found between the experimentally determined and calculated pI values of peptides.

Separation of peptides on Empore thin-layer chromatography sheets and blotting onto polyvinylidene difluoride membranes with subsequent gas phase sequencing.

Methods for the separation of peptides on a new type of thin-layer chromatography (TLC) sheet and blotting onto polyvinylidene difluoride (PVDF) membranes with subsequent gas phase sequencing are described. For validation, the A and B chain of insulin were chromatographed on Empore TLC sheets and either extracted or blotted onto PVDF membranes. The advantages and disadvantages of thin-layer chromatography on Empore sheets versus high performance liquid chromatography (HPLC) are discussed, along with the possibility of combining the two methods. In addition, TLC was combined with electrophoresis (fingerprinting) for the separation of complex peptide mixtures. Blotting from TLC sheets onto PVDF membranes was performed in two ways: contact diffusion and electrophoretic transfer. In our experiments electroblotting was more effective. Amino acid sequence determination of the B chain of insulin was possible both after extraction from the TLC sheet and after blotting onto PVDF membranes. In the former case, liquid phase sequencing and, in the latter case, gas phase sequencing was performed. The possibility to blot from TLC sheets onto membranes, e.g. PVDF, may prove useful in many fields, for example in biochemistry, and in molecular and cell biology.

Automated high-resolution two-dimensional liquid chromatographic system for the rapid and sensitive separation of complex peptide mixtures

An automated two-dimensional liquid chromatographic system for the rapid and sensitive separation of complex peptide mixtures is presented. The method presents an application of the column-switching technique, and performs sequential anion-exchange and reversed-phase chromatography under a programme directed by a computer-assisted controller. To facilitate rapid and sensitive separations, short analytical columns (3.5 cm in length) packed with non-porous packing materials of small particle size (2.5 μm) were selected for both dimensional separations, and the dead volumes of the flow system were reduced to the minimum. With this system, complex peptide mixtures such as a crude peptide fraction prepared from brain extracts were resolved into ca. 150 peaks within 80 min, with a detection limit of 10 ng. The method can be used for the systematic analysis of biologically active peptides and for the micro-scale separation of peptide fragments in the strategy of protein and gene sequence analysis.

Determination of gradient elution conditions for the separation of peptide mixtures by reversed-phase high-performance liquid chromatography: bovine β-casein tryptic digest

SummaryTaking as an example a tryptic hydrolysate of bovine β-casein, it was shown that the method used for the determination of optimal elution conditions for isocratic systems also applied to the separation of complex peptide mixtures by reversed-phase high-performance liquid chromatography with linear gradients. Using a C18 column and the same solvents, successive studies of the influence of flow rate, pH and temperature allowed a satisfactory separation of the sample in less than 30 min. Valuable information on the specificity of the action of trypsin on β-casein was deduced from the yield of the eluted peptides.

18] High-pressure liquid chromatography and radioimmunoassay for the specific and quantitative determination of endorphins and related peptides

Publisher Summary This chapter discusses high-pressure liquid chromatography (HPLC) and radioimmunoassay for the specific and quantitative determination of endorphins and related peptides. Several studies have shown that reversed-phase HPLC systems can be very useful for the separation of complex peptide mixtures. Retention on reversed-phase columns, which are nonpolar, is highly dependent on both molecular weight and polarity of the applied compounds. In general, for peptides retention, times appear to be proportional to their chain lengths and inversely proportional to their polarities as indicated by the solubilities of the constituent amino acids in water. The high resolving power of the HPLC system used allows the separation of peptides that differ in only one amino acid residue. The retention time enhancing effect of the introduction of apolar leucine as the C-terminus of the peptide sequence is remarkable. A potentially serious problem is the low recovery from a μBondapak C18 column after applying subnanogram amounts of peptides. Addition of glycylglycine to both the mobile phases at a final concentration of 1.5 × 10 −5 M can overcome this problem without interfering with the reversed-phase HPLC separation of enkephalins and endorphins, and will lead to almost complete recovery after the injection of peptide mixtures in picogram amounts.