Development of a monolithic silica extraction tip for the analysis of proteins

In proteomics, pre-treatment of sample is the most important procedure to remove the matrix for interfacing with mass spectrometry (MS). Additionally, for the samples with low concentration, the process of pre-concentration is required before MS analysis. We have newly developed solid-phase extraction (SPE) tool with pipette-tip shape for purification of bio-samples of various characteristics, utilizing monolithic silica gel as medium. The monolithic silica surface was modified with a C18 phase or coated with titania phase. A C18-bonded tip and a non-modified tip were used for sample concentration, desaltination and removal of detergents from sample. A titania-coated tip was also applied for purification and concentration of phosphorylated peptides. This novel pre-treatment method using monolithic silica extraction tip is much effective and suitable for protein analysis.     

Selective isolation-detection of two different positively charged peptides groups by strong cation exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry: application to proteomics studies

We report here a procedure for the independent analysis of two groups of peptides by liquid chromatography-matrix-assisted laser desorption/ionization mass spectrometry (LC-MALDI MS/MS), using a selective isolation-detection procedure. In this procedure all primary amino groups of tryptic peptides derived from mouse liver proteins are blocked, restricting their positive charge, at acidic pH, to the presence of histidine and arginine residues. After strong cation exchange chromatography, multiply charged peptides (R + H > 1) are retained on the column and separated with high selectivity from singly (R + H = 1) and neutral peptides (R + H = 0) which are together collected in the flow-through. Using LC-MALDI-MS/MS analysis, the retained fraction displayed a 94% of enrichment of multiply charged peptides while in the flow-through; peptides with at least one arginine or histidine residue were exclusively identified, which suggests that MS detection in this fraction is restricted only to those peptides with ionizable side chains, arginine and histidine amino acids.     

流动注射-分光光度-原子吸收串联技术在硅酸盐分析中的应用研究

本文利用流动注射、分光光度、原子吸收联测技术,对硅酸盐系统分析中的Mg、Na、P和Ti进行了Ti-Mg和P-Na联测的技术探讨。装置简单,操作方便,快速。方法用于地质矿产部一级岩石标准参考样,分析结果与推荐值吻合。     

Determination of calcium ions tightly bound to proteins.

A rapid and sensitive procedure is described for the determination of calcium ions tightly bound to proteins using high-performance gel filtration chromatography, followed by the destabilization of the protein conformation and fluorimetric analysis with Quin-2. With this method, contaminating, unbound calcium can be eliminated simultaneously and one can determine the content of a calcium ion in a protein utilizing less than 200 pmol of the protein.     

Protein binding of coherin and other small molecules by thin-layer gel chromatography

The protein binding stoichiometry of small molecules is here determined on a nanomole scale by a simplified procedure utilizing chromatography on thin layers of cross-linked dextran gels. New data are presented on the thin layer chromatographic properties of representative ligands, including a-amino acids, peptides, dyes and fluorigenic reagents, in relation to their molecular weights, polar characteristics, gel water regain values and denaturants, providing criteria for the general application of this method to studies of ligand binding with large as well as small molecules. By this procedure coherin peptides, A1 and B1--4, respectively, bind to coherin C in the molar ratio, 2:1, with a binding constant of about 10(5) M-1. Coherin C is believed to act as a carrier peptide.     

Mass spectrometric sequencing of individual peptides from combinatorial libraries via specific generation of chain-terminated sequences

Combinatorial peptide libraries are a versatile tool for drug discovery. On-bead assays identify reactive peptides by enzyme-catalyzed staining and, usually, sequencing by Edman degradation. Unfortunately, the latter method is expensive and time-consuming and requires free N termini of the peptides. A method of rapid and unambiguous peptide sequencing by utilizing synthesis-implemented generation of termination sequences with subsequent matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis is introduced here. The required capped sequences are determined and optimized for a specific peptide library by a computer algorithm implemented in the program Biblio. A total of 99.7% of the sequences of a heptapeptide library sample could be decoded utilizing a single bead for each spectrum. To synthesize these libraries, an optimized capping approach has been introduced.     

Purification of Radioiodinated Peptides with PRP-1 Polystyrene Cartridges and HPLC: Application to Atrial Natriuretic Factor and Vasopressin

Abstract

A simple and rapid cleanup procedure is described for the purification of iodinated peptides using PRP-1 polystyrene cartridges following the radioiodination process. The method is validated using different volumes and solvent systems and compared to the standard Sep-Pak C₁₈ procedure. In this study, the method is used to prepare ¹²⁵I-labeled atrial natriuretic factor and arginine-vasopressin which are further purified by reverse phase HPLC giving maximally obtainable specific activity required for the radioimmunoassays of these peptides.     

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.     

Matrix-assisted laser desorption/ionization on-target method for the investigation of oligosaccharides and glycosylation sites in glycopeptides and glycoproteins

The significance of glycoproteins in living systems instigates the ceaseless expansion of new techniques and procedures for the analysis of biological samples. Many of these applications are focused on improving the detection limit of analyzed material. In a previous study, we described a procedure for the detection of oligosaccharides cleaved from tryptic glycopeptides. Treatment of deglycosylated fractions with phenylhydrazine gave rise to peaks consistent with labeled glycans, and both types of compounds--deglycosylated peptides and oligosaccharides--were recorded from one spot and observed in one matrix-assisted laser desorption/ionization (MALDI) mass spectrum for the first time. Here, we added an additional step to this simple procedure of deglycosylating glycopeptides directly from the target spot of the first analyzed glycosylated peptides. For the purpose of this new study, a mixture of 2-aza-2-thiothymine and phenylhydrazine hydrochloride showed to be an excellent matrix for glycopeptides, oligosaccharides, deglycosylated peptides and moreover it allowed PNGaseF to be active enough to cleave oligosaccharides from peptides. The efficiency of this procedure is demonstrated on a series of intact glycoproteins and on the analysis of tryptic peptides obtained from IgG and total mouse serum. This one-step on-target deglycosylation method with subsequent derivatization on the same spot makes MALDI-MS analyses of glycopeptides fast, simple and accessible for biological samples, where classical procedures cannot produce useful results.     

An efficient procedure for extraction and determination of steroids in the tissue of laboratory animals

Summary A high-performance liquid chromatographic method for analysis of steroids in the tissue of laboratory animals is described. A two-stage solid extraction procedure, utilizing Sep-pak C₁₈ and graphitized carbon black, gives a purified product. Steroids in this extract were separated by multistep gradient elution with a reversedphase liquid chromatography system, using acetonitrilewater as solvent. Several columns with different geometry and phase loading were investigated. Optimum resolution was obtained with a medium covered and spherical particle.The applicability of this method was successfully demonstrated in the analysis of the testis and adrenal gland of rat.Dedicated to Prof. Dr. A. Liberti on the occasion of his 70th birthday.     

Identification and assay of phosphoserine and tyrosine-O-sulphate in fibrinopeptides by reversed-phase high-performance liquid chromatography

A procedure utilizing reversed-phase high-performance liquid chromatography (HPLC) is described for the identification and quantitation of individual phosphorylated and sulphated fibrinopeptides present in fibrin clot supernatants. Fibrinopeptides from human, rabbit and canine fibrinogens, which have different structures and degrees of phosphorylation and sulphation, were used to demonstrate the applicability of these methods. The procedure relies on the increased peptide hydrophobicity following removal of highly charged phosphate or sulphate groups. Dephosphorylated or desulphated peptides are thus more strongly retained on the reversed-phase HPLC column and are eluted later than their corresponding phosphorylated or sulphated peptide counterparts. Dephosphorylation is achieved by treatment of fibrinopeptide-containing clot supernatants with alkaline phosphatase. Phosphorylated peptides are characterized by an increased retention time resulting from loss of phosphate, whereas non-phosphorylated peptides remain unaffected. Similarly, a prolongation of the peptide retention time resulting from desulphation by mild acid hydrolysis serves to verify sulphation of a peptide.     

Comparison between transient isotachophoretic capillary zone electrophoresis and reversed-phase liquid chromatography for the determination of peptides in plasma.

Low levels of peptide drugs in human plasma can be determined employing off-line solid-phase extraction, followed by capillary zone electrophoresis with UV detection. A bioanalytical procedure is presented, using gonadorelin and angiotensin II in human plasma as model compounds. The solid-phase extraction method, based on a weak cation exchange mechanism, is able to remove interfering endogenous components from the plasma sample, extract the model peptides quantitatively, and give a possibility of concentrating the sample at the same time. Transient isotachophoretic conditions were kept to increase the sample loadability by about two orders of magnitude. Up to about 70% of the capillary was filled with the reconstituted extract, whereafter the peptides were selectively concentrated during the first 15 min. Subsequently, the concentrated sample zones were separated under capillary zone electrophoresis conditions, showing the technique's high resolution. For the model cationic peptides (gonadorelin, angiotensin II) good linearity and reproducibility was observed in the 20-100 ng/mL concentration range. A more extensive washing procedure permits quantitation of gonadorelin at the 5 ng/mL level. In comparison with a liquid chromatography analysis, superior mass sensitivity and separation are obtained with the transient isotachophoretic capillary zone electrophoresis method. Moreover, in this case equivalent sensitivity is achieved when it is directly compared with a liquid chromatography method with UV detection, keeping in mind that 60 times more sample is needed for the latter method. A further gain in sensitivity can be obtained when the analysis is combined with native fluorescence detection, as is demonstrated by combining liquid chromatography separation with fluorescence detection.     

Hydrophilic properties as a new contribution for computer-aided identification of short peptides in complex mixtures

A new method to predict elementary amino acid (AA) composition of peptides (molar mass <1,000 g/mol) is described. This procedure is based on a computer-aided method using three combined analyses-reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and capillary electrophoresis coupled with mass spectrometry-and using a software calculating all possible amino acid combinations from the mass of any given peptide. The complementarity between HILIC and RPLC was demonstrated. Peptide retention prediction in HILIC was successfully modelled, and the achieved prediction accuracy was as high as r2=0.97. This mathematical model, based on amino acid retention contributions and peptide length, provided the information about peptide hydrophilicity that was not redundant with its hydrophobicity. Correlations between respectively the hydrophobicity coefficients and RPLC retention time, hydrophilicity and HILIC retention time, and electrophoretic mobility and migration time were used for ranking all potential AA combinations corresponding to the given mass. The essential contribution of HILIC in this identification strategy and the need to combine the three models to significantly increase identification capabilities were both shown. Applied to an 18-standard peptide mixture, the identification procedure enabled the actual AA combination determination of the 14 di- to pentapeptides, in addition to an over 98 % reduction of possible combination numbers for the four hexapeptides. This procedure was then applied to the identification of 24 unknown peptides in a rapeseed protein hydrolysate. The effective AA composition was found for ten peptides, whereas for the 14 other peptides, the number of possible combinations was reduced by over 95 % thanks to the association of the three analyses. Finally, as a result of the information provided by the analytical techniques about peptides present in the mixture, the proposed method could become a highly valuable tool to recover bioactive peptides from undefined protein hydrolysates.     

Detergent‐assisted sample preparation for MALDI‐MS: Investigation of octylglucoside and docecylmaltoside for matrix crystallization,on‐plate digestion,and trypsin activity

We show an easy and fast method for improved detection of lipophilic peptides with MALDI‐MS utilizing the nonionic detergents n‐octylglucoside and n‐dodecylmaltoside (laurylmaltoside). Investigations comprised on‐plate digestion of proteins with trypsin, detergent effects on the protease trypsin, and the changes in MALDI matrix crystallization. Investigations also exhibited a higher tryptic activity in trypsin activity assay of 139% when using laurylmaltoside as supplement. Crystallization changed toward a more homogeneous crystal distribution and especially trypsinized insulin spectra recorded with MALDI‐MS showed improved detectability of lipophilic peptides.     

Towards the Chemical Synthesis of Proteins

The chemical total synthesis of proteins using solid supports has made great progress. It is therefore becoming a reality what E. Fischer already predicted in 1902 1 1 Cited from Nobel Lectures—Chemistry 1901-1921, Elsevier, Amsterdam 1966, p. 34.
: “I foresee the time when physiological chemistry… is able to prepare synthetic enzymes.” The synthesis of peptides of up to 20 amino acids by the well-established solid-phase procedure on polymeric supports (SPPS) has made great progress through the development of newer supports. At the same time, methods have emerged which facilitate the separation and characterization of peptides, thus allowing optimization of the synthesis of pure materials. The new supports which enable one to synthesize peptides by a rapid continuous flow procedure are characterized by hydrophilicity, beads of approximately equal size, similar swelling properties in the solvents used for peptide synthesis, and stability at high pressure. With graft copolymers of weakly cross-linked polystyrene and linear polyethylene glycol (PEG), the synthetic cycle for coupling of one amino acid can routinely be reduced to 10–20 min with a concomitant higher yield. With beads of monodispersed graft copolymer of 10 μm diameter, a synthetic cycle can, in principle, be shortened to 1–5 min. By utilizing this high-speed solid-phase procedure, larger peptides up to the size of proteins could also be prepared in a few hours. With newer mass spectroscopic methods such as ion-spray mass spectrometry, peptides of up to a molecular mass of 100 kDa can be characterized, and with the advent of capillary electrophoresis, another very efficient separation tool, besides HPLC, is at our disposal.     

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.     

MeCAT—new iodoacetamide reagents for metal labeling of proteins and peptides

Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metal-coded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labeling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent.     

A manual sequence method of peptides and phosphopeptides using 4-(1'-cyanoisoindolyl)phenylisothiocyanate

A method for sequence analysis and identification of phosphoamino acids in peptides based on high performance liquid chromatography (HPLC) is described. The peptides were derivatized with an Edman type reagent, 4-(1'-cyanoisoindolyl)phenylisothiocyanate (CIPIC) and subsequently cleaved to generate stable and fluorescent 4-(1'-cyanoisoindolyl)phenylthiazolinone (CIP-TZ)-amino acids. Several experimental factors that affected derivatization on membranes were examined. Under the optimized conditions, the CIP-TZ derivatives of Try(p), Thr(p) and Ser(p) were obtained and separated from their parent amino acids with baseline resolution using an isocratic elution system. Up to the 4th residue of phosphorylated pentapeptides was successfully identified, whereas phosphoamino acid residues could not be detected by the conventional procedure using phenylisothiocyanate (PITC). The results demonstrated the potential of CIPIC as a derivatization reagent for peptide sequencing and the applicability of the method for the study and identification of phosphoamino acids in peptides.     

Derivatization of Aminophosphonic Acids for HPLC Analysis

Abstract

A general method for the derivatization of alpha-aminophosphonic acids for HPLC analysis is described. The method involves the conversion of the amino functionality to the corresponding 9-fluorenylmethoxycarbonyl (FMOC) urethane followed by esterification of the phosphonic acid moiety utilizing triethyl orthoformate. The sensitivity is compared to that achievable with a previously described N-trifluoroacetyl diethyl ester procedure on the basis of UV detection. Derivative separation using fluorescence detection is also shown.     

Development of a capillary electrophoresis method for the characterization of collagens in cartilage tissue

The use of capillary electrophoresis (CE) for the separation of peptides specific to type I and type II collagen is evaluated. The aim of this work is to develop a method to characterize cartilage, cartilage repair tissue, and tissue engineered cartilage. The analysis is dependent on the cleavage of collagen into constituent peptides by cyanogen bromide. A number of these peptides are specific to the collagen type. CE is evaluated for the separation of these specific peptides using uncoated and coated capillaries over a wide range of pH and buffer concentrations. Separation of peptides specific to type I and type II collagen is achieved using a Supelco CElect N capillary and a 100mM phosphate buffer at pH 6. Meniscal cartilage is characterized using this method. The proportion of type I collagen to type II collagen corresponds well with that reported by others and indicates the potential of this method for the characterization of cartilage.