Enrichment and desalting of tryptic protein digests and the protein depletion using boron nitride

Sample preparation still remains a great challenge in modern bioanalysis and the interest in new efficient solid phase extraction (SPE) materials still remains high. In this work, hexagonal boron nitride (h-BN) is introduced as a new SPE material for the isolation and enrichment of peptides. The h-BN is isoelectronic and structurally similar to graphite. It has remarkable properties including good thermal conductivity, excellent thermal and chemical stability and a better oxidation resistance than graphite. BN attracts increasing interest because of its wide range of applicability. In the present work, the great potential of h-BN, as a new SPE-material, on the enrichment, preconcentration and desalting of tryptic digest of model proteins is demonstrated. A special attention was dedicated to the efficient enrichment of hydrophilic phosphopeptides. Two elution protocols were developed for the enrichment of peptides compatible for subsequent MALDI-MS and ESI-MS analysis. In addition, the recoveries of 5 peptides and 3 phosphopeptides with wide range of pI values utilizing h-BN materials with different surface areas were investigated. 84–106% recovery rate could be achieved using h-BN materials. The results were compared with those obtained using graphite and silica C18 under the same elution conditions, and lower recoveries were obtained. In addition, h-BN was found to have a capability of protein depletion, which is requisite for the peptide profiling.     

Improved sensitivity for insulin in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry by premixing alpha-cyano-4-hydroxycinnamic acid matrix with transferrin

This report describes an enhancement of the signal intensities of proteins and peptides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). When alpha-cyano-4-hydroxycinnamic acid (CHCA) premixed with human transferrin (Tf) was used as a matrix, the signal intensity of insulin was amplified to more than ten times that of the respective control in CHCA without Tf. The detection limit of insulin was 0.39 fmol on-probe in the presence of Tf, while it was 6.3 fmol in the absence of Tf. The signal intensity of insulin was also enhanced when the CHCA matrix was premixed with proteins other than Tf (80 kDa), such as horse ferritin (20 kDa), bovine serum albumin (BSA, 66 kDa), or human immunoglobulin G (150 kDa). The optimum spectrum of insulin was obtained when the added amount of protein was in the range 0.26-0.62 pmol, regardless of the molecular weight of the added protein. Tf and BSA outperformed the other tested proteins, as determined by improvements in the resulting spectra. When the mass spectra of several peptides and proteins were recorded in the presence of Tf or BSA, the signal intensities of large peptides such as glucagon were enhanced, though those of smaller peptides were not enhanced. In addition, the signal enhancement achieved with Tf and BSA was more pronounced for the proteins, including cytochrome C, than for the large peptides. This enhancement effect could be applied to improve the sensitivity of MALDI-TOFMS to large peptides and proteins.     

Two-stage Off-Gel isoelectric focusing: protein followed by peptide fractionation and application to proteome analysis of human plasma

This paper presents the recently introduced Off-Gel electrophoresis (OGE) technology as a versatile tool to reproducibly fractionate intact proteins and peptides into discrete liquid fractions. The coupling of two stages of OGE, i.e., the separation of intact proteins in a first-stage followed by fractionation of peptides derived from each protein fraction after proteolysis in a second stage, results in an array of 15 x 15 fractions that are directly amenable to additional peptide fractionation like reverse-phase liquid chromatography (RPC). The analysis of all second-stage peptide fractions from only the first-stage protein fraction representing pH 5.0 -5.15 by on-line reverse-phase LC-tandem mass spectrometry resulted in the identification of 53 proteins (337 peptides), of which 10 were on different immunoglobulin (Ig) chains, with an input of only 1.5 mg human blood plasma proteins. Increasing the protein load to approximately 12 mg increased the number of identified proteins in the same protein fraction to 73 proteins (449 peptides), of which 15 were Ig-related. Immunodepletion of six of the most abundant proteins (albumin, transferrin, haptoglobin, IgG, IgA, and alpha-1-antitrypsin) prior to first-stage OGE with an input of 1.5 mg of protein (equivalent to approximately 10 mg nondepleted plasma) resulted in the identification of 81 proteins (660 peptides), of which three were still Ig fragments. The pI-based separation of peptides appears to be nonuniform based on the theoretically determined pI values of identified peptides. This observation specifically accounts for the neutral zone (pI 5-8) and can be accounted for by the physicochemical properties of the peptides given by their amino acid composition. The power of OGE separation of proteins and peptides is discussed with a focus on the use of the knowledge about the pI of proteins and peptides that assist the validation of correct identifications together with the retention time of peptides on RPC.     

Electrochemistry of capillary systems with narrow pores. VI. Convection conductivity (theoretical considerations)

Generally, the electrical convection current and the electrical convection conductivity (Smoluchowski's surface conductivity) have to be taken into account to describe transport phenomena across membranes with narrow pores although the electrical charge distribution within the pores cannot be described as a Helmholtz electrical double layer. In collodion membranes, which have a comparatively low fixed ion concentration, the contribution of the convection conductivity to the electrical conductivity of the pore fluid may be neglected. This assumption was made tacitly in the analysis of our data obtained with this type of membrane.

In this communication equations are derived which take the convection conductivity into account. They are in agreement with the phenomenological transport equations developed by Staverman on the basis of the thermodynamics of irreversible processes.

The electrical convection conductivity can be considered to be the contribution of the fixed ion concentration to the electrical conductivity. It is argued that this contribution cannot be neglected in ion exchange membranes with a high fixed ion concentration and a high mechanical permeability. Neglecting the electrical convection conductivity in such systems could lead to considerable differences between experimental conductivity data and the theoretical predictions. An electrical conductivity term for the fixed ions is proposed which can be used as a correction factor in the equations in which the contribution of the electrical convection conductivity has been neglected. Suggestions are made for the measurement of the electrical convection conductivity in systems with narrow pores and high electrical conductivity (e.g. ion exchange resins). The consequences of the electrical convection conductivity in practical applications of ion-exchange resins are discussed (acceleration of the rates of ion exchange; improvement of the separation properties by the application of a direct electrical current flow).     

Electrochemistry of capillary systems with narrow pores. VI. Convection conductivity (theoretical considerations)

Generally, the electrical convection current and the electrical convection conductivity (Smoluchowski's surface conductivity) have to be taken into account to describe transport phenomena across membranes with narrow pores although the electrical charge distribution within the pores cannot be described as a Helmholtz electrical double layer. In collodion membranes, which have a comparatively low fixed ion concentration, the contribution of the convection conductivity to the electrical conductivity of the pore fluid may be neglected. This assumption was made tacitly in the analysis of our data obtained with this type of membrane.In this communication equations are derived which take the convection conductivity into account. They are in agreement with the phenomenological transport equations developed by Staverman on the basis of the thermodynamics of irreversible processes.The electrical convection conductivity can be considered to be the contribution of the fixed ion concentration to the electrical conductivity. It is argued that this contribution cannot be neglected in ion exchange membranes with a high fixed ion concentration and a high mechanical permeability. Neglecting the electrical convection conductivity in such systems could lead to considerable differences between experimental conductivity data and the theoretical predictions. An electrical conductivity term for the fixed ions is proposed which can be used as a correction factor in the equations in which the contribution of the electrical convection conductivity has been neglected. Suggestions are made for the measurement of the electrical convection conductivity in systems with narrow pores and high electrical conductivity (e.g. ion exchange resins). The consequences of the electrical convection conductivity in practical applications of ion-exchange resins are discussed (acceleration of the rates of ion exchange; improvement of the separation properties by the application of a direct electrical current flow).     

Combination of RSM and NSGA-II algorithm for optimization and prediction of thermal conductivity and viscosity of bioglycol/water mixture containing SiO2 nanoparticles

The fluids containing nanoparticles have enhanced thermo-physical characteristics in comparison with conventional fluids without nanoparticles. Thermal conductivity and viscosity are thermo-physical properties that strongly determine heat transfer and momentum. In this study, the response surface method was firstly used to derive an equation for the thermal conductivity and another one for the viscosity of bioglycol/water mixture (20:80) containing silicon dioxide nanoparticles as a function of temperature as well as the volume fraction of silicon dioxide. Then, NSGA-II algorithm was used for the optimization and maximizing thermal conductivity and minimizing the nanofluid viscosity. Different fronts were implemented and 20th iteration number was selected as Pareto front. The highest thermal conductivity (0.576 W/m.K) and the lowest viscosity (0.61 mPa.s) were obtained at temperature on volume concentration of (80 °C and 2%) and (80 °C without nanoparticle) respectively. It was concluded that the optimum thermal conductivity and viscosity of nanofluid could be obtained at maximum temperature (80 °C) or a temperature close to this temperature. An increase in the volume fraction of silicon dioxide led to the enhancement of thermal conductivity but the solution viscosity was also increased. Therefore, the optimum point should be selected based on the system requirement.     

Studies on the primary structure of cow kappa-casein. Structural features of para-kappa-casein; N-terminal sequence of kappa-caseinoglycopeptide studied with a sequencer

Several long tryptic peptides obtained from reduced maleylated χ-casein were sequenced: they belonged to the N- and C-terminal (37 residues) moieties of para-χ-casein. Several tryptic peptides could then be joined by chymotryptic overlap peptides. 102 amino acid residues of para-χ-casein were thus placed into two long and four shorter sequences. Some structural results were established with a Sequencer which was also employed for a verification of the N-terminal sequence of the χ-caseinoglycopeptide; a previously described short glycopeptide was found again and was reinvestigated.     

Easy amino acid sequencing of sulfonated peptides using post-source decay on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer equipped with a variable voltage reflector

Tryptic peptides were labeled with sulfonic acid groups at the N-termini using an improved chemistry. The derivatization was performed in common aqueous buffers on peptides adsorbed onto a ZipTip trade mark C(18), thus allowing simultaneous desalting/concentration of the sample. When only Arg-terminating peptides were considered, the procedure from adsorption onto the ZipTip until analysis by MALDI-PSD took about 10 min and several samples could be worked on in parallel. The resulting improved post-source decay (PSD) fragmentation produced spectra containing only y-ions. PSD amino acid sequencing of underivatized and derivatized synthetic peptides was compared. From the sequence information obtained from derivatized peptides isolated by ion selection from tryptic in-gel digests, a protein was correctly identified which was difficult to analyze from an unclear peptide mass fingerprint analysis. The method was also applied to the identification and localization of phosphorylated Ser and Tyr residues in native and synthetic peptides.     

酶法获得的多肽库用于新型手性固定相的制备

采用胰蛋白酶酶解牛血清白蛋白,将制备的酶解液超滤,得到相对分子质量小于6000的小分子多肽库。应用羰基咪唑法将该多肽库键合至多孔硅胶,得到一种新型手性固定相。应用该新型手性固定相成功地拆分了两种氨基酸对映体。     

样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响

人乳内源肽是乳蛋白在乳腺中被降解形成的具有生理功能的肽,是人乳的重要组成部分,研究人乳内源肽对于婴儿健康具有重要的意义.高效液相色谱-串联质谱(LC-MS/MS)联用技术的应用,促使人乳内源肽的研究取得了突破性的进展.人乳中内源肽含量低、干扰组分多,样品制备方法是影响分析结果的关键步骤.为了研究样品制备方法对分析结果的...     

Selection of possible marker peptides for the detection of major ruminant milk proteins in food by liquid chromatography-tandem mass spectrometry

The aim of this work was the determination of peptides, which can function as markers for identification of milk allergens in food samples. Emphasis was placed on two casein proteins (α- and β-casein) and two whey proteins (α-lactalbumin and β-lactoglobulin). In silico tryptic digestion provided preliminary information about the expected peptides. After tryptic digestion of four milk allergens, the analytical data obtained by combination of reversed-phase high performance liquid chromatography and quadrupole tandem mass spectrometry (LC-MS/MS) led to the identification of 26 peptides. Seven of these peptides were synthesized and used for calibration of the LC-MS/MS system. Species specificity of the selected peptides was sought by BLAST search. Among the selected peptides, only LIVTQTMK from β-lactoglobulin (m/z 467.6, charge 2+) was found to be cow milk specific and could function as a marker. Two other peptides, FFVAPFPEVFGK from α-casein (m/z 693.3, charge 2+) and GPFPIIV from β-casein (m/z 742.5, charge 1+), occur in water buffalo milk too. The other four peptides appear in the milk of other species also and can be used as markers for ruminant species milk. Using these seven peptides, a multianalyte MS-based method was developed. For the establishment of the method, it was applied at first to different dairy samples, and then to chocolate and blank samples, and the peptides could be determined down to 1 ng/mL in food samples. At the end, spiked samples were measured, where the target peptides could be detected with a high recovery (over 50%).     

High-performance liquid chromatography with contactless conductivity detection for the determination of peptides and proteins using a monolithic capillary column

Gradient programs were applied to the determination of peptides and proteins in HPLC with contactless conductivity detection. A monolithic capillary column was used for the fast and sensitive determination of the biochemical species in acidic mobile phases consisting of acetic acid or trifluoroacetic acid in various concentrations of acetonitrile in water. The drift in baseline, which is caused by conductivity changes during the elution program, was minimized by careful optimization of the composition of the mobile phase and remaining drift was removed by computational baseline normalization. The flow rate from a conventional HPLC pump was reduced to a flow rate suitable for capillary systems using a pre-column flow splitter and a final total flow rate of 1.65 microl/min was used for all capillary HPLC separations. The contactless conductivity detector was positioned directly on the outlet capillary of the separation column and positively charged peptides and proteins were determined as sharp and symmetrical peaks. Detection limits in a concentration range from 3.7 x 10(-8) to 5.1 x 10(-7)M and a reproducibility of peak areas and peak heights between 2.3% and 7.3% were achieved for all biochemical species tested.     

Synthesis of cleavable peptides with authentic C-termini: an application for fully automated SPOT synthesis

A simple method for the synthesis of carboxyl-free peptides on cellulose membranes was improved and adapted for fully automated SPOT synthesis. Using 1,1′-carbonyl-di-imidazole (CDI) or 1,1′-carbonyl-di-(1,2,4-triazole) (CDT) as an activator within a defined period of time, we were able to reduce the formation of di- or oligomerization of the C-terminal amino acid. The soluble peptides are obtained in a purity range of 60-95% and could be used directly for different biological assays (e.g., CD8 T-cell epitope) that require authentic C-termini.     

Novel phosphoryl derivatization method for peptide sequencing by electrospray ionization mass spectrometry

A one-step phosphoryl derivatization method has been used in a peptide sequencing procedure for electrospray ionization tandem mass spectrometry (ESI-MS/MS). The sodiated derivatized peptides exhibit very simple dissociation patterns, in which two kinds of fragment ions, [b(n) + OH + Na]+ and [a(n) + Na]+, are formed. Since the amino acid residues are lost sequentially from the C-terminus, peptide sequences can be identified easily. The fragmentation efficiency of peptides increased as a result of the phosphorylation, and also provided peaks of useful intensity at lower m/z. A peptide with lysine at the C-terminus was derivatized and analyzed by ESI-MS/MS. Similar mass spectra, from which the sequence could be read out, were obtained. This is a novel derivatization method yielding neutral derivatives that should be suitable for peptide sequencing by LC/ESI-MS/MS.     

Comparison of silica-based cyanopropyl and octyl reversed-phase packings for the separation of peptides and proteins.

The performance of a silica-based C8 packing was compared with that of a less hydrophobic, silica-based cyanopropyl (CN) packing during their application to reversed-phase high-performance liquid chromatography (linear trifluoroacetic acid-water to trifluoroacetic acid-acetonitrile gradients) of peptides and proteins. It was found that: (1) the CN column showed excellent selectivity for peptides which varied widely in hydrophobicity and peptide chain length; (2) peptides which could not be resolved easily on the C8 column were widely separated on the CN column; (3) certain mixtures of peptides and small organic molecules which could not be resolved on the C8 column were completely separated on the CN column; (4) impurities arising from solid-phase peptide synthesis were resolved by a wide margin on the CN column, unlike on the C8 column, where these compounds were eluted very close to the peptide product of interest: and (5) specific protein mixtures exhibited superior resolution and peak shape on the CN column compared with the C8 column. The results clearly demonstrate the effectiveness of employing stationary phases of different selectivities (as opposed to the more common optimization protocol of manipulating the mobile phase) for specific peptide and protein applications, an approach underestimated in the past.     

Simultaneous separation and quantitation of the major bovine whey proteins including proteose peptone and caseinomacropeptide by reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene

A precise, sensitive and reliable RP-HPLC method was developed to enable not only unequivocal determination of alpha-lactalbumin and beta-lactoglobulin in bovine whey samples, but also simultaneous measurement of proteose peptone, caseinomacropeptide, bovine serum albumin and immunoglobulin G. The optimised method on the Resource RPC column allowed separation of the proteins in 30 min and could be applied to the analysis of soluble proteins in a variety of commercial and laboratory whey products. Furthermore, some qualitative information on protein heterogeneity and quality could be derived from the RP-HPLC analyses with additional data available from on-line electrospray mass spectrometry. Within- and between-day repeatability over a wide range of concentrations was excellent (RSD< or =5%) for all proteins except immunoglobulin G and bovine serum albumin where RSD was 7-10%. Analysis of grouped data from whey protein concentrate and whey protein isolate samples gave a limit of detection of < or =0.3% powder mass and a limit of quantitation of < or =1.0% powder mass for all proteins except immunoglobulin G. Limits of detection and quantitation were 0.6% and 2.0%, respectively, for this protein. Quantitative data obtained by the RP-HPLC method compared very favourably with data obtained by alternative methods of whey protein analysis.     

Ribosomal synthesis of N-methyl peptides

N-methyl amino acids (N-Me AAs) are a common component of nonribosomal peptides (NRPs), a class of natural products from which many clinically important therapeutics are obtained. N-Me AAs confer peptides with increased conformational rigidity, membrane permeability, and protease resistance. Hence, these analogues are highly desirable building blocks in the ribosomal synthesis of unnatural peptide libraries, from which functional, NRP-like molecules may be identified. By supplementing a reconstituted Escherichia coli translation system with specifically aminoacylated total tRNA that has been chemically methylated, we have identified three N-Me AAs (N-Me Leu, N-Me Thr, and N-Me Val) that are efficiently incorporated into peptides by the ribosome. Moreover, we have demonstrated the synthesis of peptides containing up to three N-Me AAs, a number comparable to that found in many NRP drugs. With improved incorporation efficiency and translational fidelity, it may be possible to synthesize combinatorial libraries of peptides that contain multiple N-Me AAs. Such libraries could be subjected to in vitro selection methods to identify drug-like, high-affinity ligands for protein targets of interest.     

Contactless conductivity detection of synthetic polymers in non-aqueous size-exclusion electrokinetic chromatography

A capacitively coupled contactless conductivity detection (CCD) system has been applied for the detection of neutral synthetic polymers in capillary size-exclusion electrokinetic chromatography (SEEC). Polystyrene standards, that were used as a model compounds, were separated on a capillary column packed with porous 10 microm silica particles with an electrokinetically driven mobile phase, and detected by CCD and UV detection simultaneously. Mass-calibration curves for polystyrene were constructed. Satisfactory results were obtained for the linearity, the run-to-run repeatability (<0.2% for the relative retention and <4% for the peak area) and the robustness of the detector. One of the major issues in this preliminary study was to investigate the origin of the peaks observed for the polystyrene standards. The effect of the molar mass of the polystyrenes on the sensitivity was small. Therefore, the signals obtained could not be explained as the result of an increased viscosity and a decreased solution conductivity of the solute zone. An alternative hypothesis is suggested, and recommendations for further research are given.     

Anion-exchange capillary electrochromatography with indirect UV and direct contactless conductivity detection

Conductivity detection is applied to ion-exchange capillary electrochromatography (IE-CEC) with a packed stationary phase, using a capacitively coupled contactless conductivity detector with detection occurring through the packed bed. Columns were packed with a polymeric latex-agglomerate anion-exchanger (Dionex AS9-SC). A systematic approach was used to determine suitable eluants for IE-CEC separations using simultaneous indirect UV and direct conductivity detection. Salicylate and p-toluenesulfonate were identified as potential eluant competing anions having sufficient eluotropic strength to induce changes in separation selectivity, but salicylate was found to be unsuitable with regard to baseline stability. It was also found for both indirect UV and direct conductivity detection that homogenous column packing was imperative, and monitoring of the baseline could be used to assess the homogeneity of the packed bed. Using a p-toluenesulfonate eluant, the separation of eight common anions was achieved in 2.5 min. Direct conductivity detection was found to be superior to indirect UV detection with regard to both baseline stability and detection sensitivity with detection limits of 4-25 microg/L being obtained. However, the calibration for each anion was not linear over more than one order of magnitude. When using conductivity detection, the concentration of the eluant could be varied over a wider range (2.5-50 mM p-toluenesulfonate) than was the case with indirect UV detection (2.5-10 mM), thereby allowing greater changes in separation selectivity to be achieved. By varying the concentration of p-toluenesulfonate in the eluant, the separation selectivity could be manipulated from being predominantly ion-exchange in nature (2.5 mM) to predominantly electrophoretic in nature (50 mM).     

Evaluation of protein quantification using standard peptides containing single conservative amino acid replacements

Structural analogs are evaluated as peptide internal standards for protein quantification with liquid chromatography‐multiple reaction monitoring mass spectrometry (LC‐MRM); specifically, single conservative amino acid replacements (SCAR) are performed to create tagged standards that differ by the addition or subtraction of a single methylene group in one amino acid side chain. Because the performance of stable isotope‐labeled standards (SIS) has been shown to be superior to structural analogs, differences in both development and quantitative performance between assays based on SIS and SCAR peptides are explored. To establish an assay using the structural analogs, analysis of endogenous, SCAR and SIS peptides was performed to examine their ion signal, fragmentation patterns and response in LC‐MRM. Performance of SCAR and SIS peptides was compared for quantification of epidermal growth factor receptor from lung cancer cell lysates and immunoglobulin M in the serum of multiple myeloma patients. Copyright © 2012 John Wiley & Sons, Ltd.