Contributions of commercial sorbents to the selectivity in immobilized metal affinity chromatography with Cu(II)

Immobilized copper(II) affinity chromatography [Cu(II)-immobilized metal affinity chromatography (IMAC)] has been used in proteomics to simplify sample mixtures by selecting histidine-containing peptides from proteolytic digests. This paper examines the specificity of four different support materials with an iminodiacetic acid (IDA) stationary phase in the selection of only histidine-containing peptides in the single step capture-release mode. Three of the sorbents examined were commercially available: HiTrap Chelating HP (agarose), TSK Chelate-5PW, and Poros 20MC. IDA was also immobilized on CIM discs (monolithic glycidylmethacrylate-ethylene dimethacrylate). Tryptic digests of transferrin and beta-galactosidase were used as model samples to evaluate these sorbents. It was found that among the examined matrices, the TSK Chelate-5PW sorbent bound histidine-containing peptides the strongest, while Poros matrix was found to have a high degree of non-specific bindings. Agarose-based columns showed relatively high selectivity and specificity.     

Automated signature peptide approach for proteomics

This paper addresses the issue of automating the multidimensional chromatographic, signature peptide approach to proteomics. Peptides were automatically reduced and alkylated in the autosampler of the instrument. Trypsin digestion of all proteins in the sample was then executed on an immobilized enzyme column and the digest directly transferred to an affinity chromatography column. Although a wide variety of affinity columns may be used, the specific column used in this case was a Ga(III) loaded immobilized metal affinity chromatography (IMAC) column. Ga(III)-IMAC is known to select phosphorylated peptides. Phosphorylated peptides selected by the affinity column from tryptic digests of milk were automatically transferred to a reversed-phase liquid chromatography (RPLC) column. Further fractionation of tryptic peptides on the RPLC column was achieved with linear solvent gradient elution. Effluent from the RPLC column was electrosprayed into a time-of-flight mass spectrometer. The entire process was controlled by software in the liquid chromatograph. With slight modification, it is possible to add multiple columns in parallel at any of the single column positions to further increase throughput. Total analysis time in the tandem column mode of operation was under 2 h.     

Quantitative proteomics strategy involving the selection of peptides containing both cysteine and histidine from tryptic digests of cell lysates

This paper describes a procedure for quantitative proteomics that selects peptides containing both cysteine and histidine residues from tryptic digests of cell lysates. Cysteine-containing peptides were selected first by covalent chromatography using thiol disulfide exchange. Following the release of cysteine-containing peptides from the covalent chromatography column with reductive cleavage, histidine-containing peptides were captured by passage through an immobilized metal affinity chromatography column loaded with copper. Quantification was achieved in a four-step process involving (i) differential labeling of control and experimental samples with isotopically differing forms of succinic anhydride, (ii) mixing the two globally labeled samples, (iii) fractionating the labeled peptides by reversed-phase liquid chromatography, and (iv) determining the isotope ratio in individual peptides by mass spectrometry. The results of these studies indicate that by selecting peptides containing both cysteine and histidine, the complexity of protein digests could be substantially reduced. Up-regulated proteins from plasmid bearing Escherichia coli that had been induced with isopropyl beta-thiogalacto-pyranoside were identified and quantified by the global internal standard technology (GIST) described above. Database searches were greatly simplified because the number of possible peptide candidates was reduced more than 95%.     

Characterization of metal affinity of green fluorescent protein and its purification through salt promoted, immobilized metal affinity chromatography

Immobilized metal affinity chromatography (IMAC) was investigated as a method of recovery for green fluorescent protein (GFPuv). It was found that in the absence of genetic modification to enhance metal affinity, GFPuv displayed strong metal affinity to Cu(II) and Ni(II), and weak or negligible affinity to Zn(II) and Co(II). Changes in the mobile phase NaCl concentration during Ni(II)-IMAC strongly affected purity and yield of GFPuv, with fine resolution under higher NaCl concentrations. Finally, IMAC via Cu(II) and Zn(II) with intervening diafiltration was used to recover GFPuv with high yield and purity.     

Simplification of complex peptide mixtures for proteomic analysis: reversible biotinylation of cysteinyl peptides

A rapid means of identifying many components in an enriched mixture of proteins is enzymatic digestion of the entire protein fraction. This complex peptide mixture is then subjected to reversed-phase high performance liquid chromatography (HPLC) coupled on-line with a mass spectrometer capable of data-dependent ion selection for fragmentation (LC-tandem mass spectrometry; MS/MS). Thus, as many peptides as possible in the sample are fragmented to produce MS/MS spectra, which can then be searched against sequence databases. Ideally, one peptide from each protein in the mixture would be fragmented and identified. To this end, we employed an affinity selection method to capture cysteinyl peptides and thereby simplify the mixture. Both the captured cysteinyl and the noncysteinyl peptides are analyzed by LC-MS/MS, to increase the number of proteins identified. The method was tested on a limited set of standard proteins and applied to the analysis of a protein fraction obtained from isolated mitochondria treated with atractyloside. To further increase the number of different precursor ions selected for fragmentation, dynamic exclusion and ion selection from multiple narrow mass ranges of consecutive runs were employed.     

Separation of human orosomucoid major gene products using immobilized copper affinity chromatography and identification of the metal-interactive residues

Summary The major gene products of human orosomucoid, GP1 and GP2, were purified using immobilized copper affinity chromatography [Cu(II)-IMAC], with 3 mM imidazole as eluent. Both gene products bound to the Cu(II)-IMAC column in the presence of 1 M NaCl, but at different pHs. GP1 was not retained after treatment with diethylpyrocarbonate (DEPC). This modification was characterized using difference absorbance spectrophotometry and mass spectrometry. The latter provided unambiguous assignment of some of the modified residues. No correlation was observed between the modification of histidine/tyrosine and protein retention. Furthermore, removal of the carbethoxy groups of modified histidine and tyrosine by hydroxylamine treatment did not improve the retention. Therefore neither histidine nor tyrosine could be the critical residues in metal recognition. Results from mass spectrometric analysis of retained and unretained fractions of DEPC modified GP 1 indicated that the lysine residues 130/135 and 152 were modified significantly in both fractions, but to a relatively less extent in the retained one. We suggest that the retnetion of GP1 involves several residues including lysines, and that a critical number of these is necessary for retention.     

Signature-peptide approach to detecting proteins in complex mixtures

The objective of the work presented in this paper was to test the concept that tryptic peptides may be used as analytical surrogates of the protein from which they were derived. Proteins in complex mixtures were digested with trypsin and classes of peptide fragments selected by affinity chromatography, lectin columns were used in this case. Affinity selected peptide mixtures were directly transferred to a high-resolution reversed-phase chromatography column and further resolved into fractions that were collected and subjected to matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The presence of specific proteins was determined by identification of signature peptides in the mass spectra. Data are also presented that suggest proteins may be quantified as their signature peptides by using isotopically labeled internal standards. Isotope ratios of peptides were determined by MALDI mass spectrometry and used to determine the concentration of a peptide relative to that of the labeled internal standard. Peptides in tryptic digests were labeled by acetylation with acetyl N-hydroxysuccinimide while internal standard peptides were labeled with the trideuteroacetylated analogue. Advantages of this approach are that (i) it is easier to separate peptides than proteins, (ii) native structure of the protein does not have to be maintained during the analysis, (iii) structural variants do not interfere and (iv) putative proteins suggested from DNA databases can be recognized by using a signature peptide probe.     

人神经母细胞瘤的磷酸化膜蛋白质组分析

针对人神经母细胞瘤SH-SY5Y细胞系的磷酸化膜蛋白质组,发展了基于多酶酶解法结合杂化硅胶基质固定化钛离子亲和色谱(Ti4+-IMAC)整体柱富集的分析策略。该方法通过对细胞裂解液进行超速离心,以及1 mol/L NaCl和0.1 mol/L Na2CO3顺序清洗,获得膜蛋白质组分。所提取的蛋白质分别经胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶平行酶解,产生的肽段经Ti4+-IMAC整体柱选择性富集磷酸肽后,采用纳升级反相液相色谱分离和质谱鉴定,成功鉴定到43个磷酸化蛋白质,其中有14个定位于膜上。研究结果表明,采用该策略开展SH-SY5Y细胞系磷酸化膜蛋白质组学分析有望加速对该肿瘤的研究和相关潜在标记物的筛选。     

Characterisation by immobilised metal ion affinity chromatographic procedures of the binding behaviour of several synthetic peptides designed to have high affinity for Cu(II) ions.

In this investigation, several peptides containing an increasing number of histidine residues have been designed and synthesised. The peptides involved repeat units of either the pentameric EAEHA or the tetrameric HLLH sequence motifs. Adsorption isotherms for these synthetic peptides and hexahistidine (hexa-His) as a control substance were measured under batch equilibrium binding conditions with an immobilised Cu(II)-iminodiacetic acid (IDA) sorbent. The experimental data were analysed in terms of Langmuirean binding behaviour. In common with previous studies with synthetic peptides, these investigations have demonstrate that the sequential organisation of the histidine side chains in these peptides can affect the selectivity of the coordination interactions with borderline metal ions in immobilised metal ion affinity chromatographic systems. The results also confirm that peptides selected on the basis of their potential to form amphipathic secondary structures with their histidine residues presented on one face of the molecule can exhibit equivalent or higher affinity constants towards copper ions than hexa-His, although they contain fewer histidine residues. These findings are thus relevant to the selection of peptides produced inter alia by combinatorial synthetic procedures to have enhanced binding properties for Cu(II) or Ni(II) ions, or intended for use as peptide tags in the fusion handle approach for the affinity chromatographic purification of recombinant proteins.     

Identification of yeast oxidized proteins: chromatographic top-down approach for identification of carbonylated, fragmented and cross-linked proteins in yeast

The effects of oxidative stress on the yeast proteome were studied using hydrogen peroxide as the stress agent. Oxidized proteins were isolated by (1) biotinylation of oxidized proteins with biotin hydrazide, (2) affinity selection using monomeric avidin affinity chromatography, and (3) further fractionated by reversed-phase liquid chromatography (RPLC) on a C(8) column. Oxidized protein fractions from RPLC were then trypsin digested and the peptide cleavage fragments identified by tandem mass spectrometry (MS/MS). Slightly over 400 proteins were identified. Sites of carbonyl formation were found in roughly one fourth of these proteins. Oxidation on other amino acids in carbonylated peptides was seen in 32 cases while carbonylation was absent in 96 of the oxidized proteins observed. Although there are large numbers of potential oxidation sites, oxidation seemed to be restricted to a small area in most of the proteins identified. Sometimes multiple amino acids in the same tryptic peptide were oxidized. A second trend was that more than 8% of the proteins identified appeared in more than one of the RPLC fractions. Based on the position of the peptides identified in the primary structure of protein candidates derived from databases it was concluded that this occurred by fragmentation of a parent protein. It is not clear from the data whether the fragmentation process was of enzymatic or oxidative origin. Finally, peptides from two or more proteins occurred together in more than one reversed phase fraction with 2% of the proteins identified. This data was interpreted to mean that this was the result of protein cross-linking.     

The resistance of metallothionein to proteolytic digestion: an LC-MS/MS analysis

Metallothioneins (MTs) are a family of cysteine-rich metalloproteins which strongly bind to heavy metals, such as Cd(II), Zn(II), and Cu(I). Previous works by other group using gel electrophoresis and fluorescence showed MTs were resistant to proteolytic digestion by a variety of enzymes, raising the difficulties in proteomic identification of MTs. The present work was attempted to analyze the resistance of MTs to trypsin using LC with MS/MS (LC-MS/MS), which was able to determine the sequences of the produced peptides and thus precisely characterize the cleavages. The results showed that metal-saturated MTs were completely resistant to trypsin. This resistance problem could be overcome by the addition of EDTA to MT samples, which rendered MTs readily digested into peptides and identified by MS/MS. Interestingly, the partially metal binding MTs were digested into peptides predominantly with miss cleavages which were well dependent on the amount of heavy metals bound to MTs. An explanation for these observations was proposed. The potential applications of the MT's resistance to trypsin in isolation and identification of MTs in complex mixtures such as cultured cells was demonstrated. The preliminary data also showed the same proteomic approach of proteolytic digestion followed by MS/MS analysis may provide information on metal binding status of MTs, along with the identification of MTs in a mixture.     

Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides

An integrated analytical strategy for enrichment, detection and sequencing of phosphorylated peptides by matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) is reported. o-Phosphoric acid was found to enhance phosphopeptide ion signals in MALDI-MS when used as the acid dopant in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. The effect was largest for multiply phosphorylated peptides, which exhibited an up to ten-fold increase in ion intensity as compared with standard sample preparation methods. The enhanced phosphopeptide response was observed during MALDI-MS analysis of several peptide mixtures derived by proteolytic digestion of phosphoproteins. Furthermore, the mixture of 2,5-DHB and o-phosphoric acid was an excellent eluant for immobilized metal affinity chromatography (IMAC). Singly and multiply phosphorylated peptide species were efficiently recovered from Fe(III)-IMAC columns, reducing sample handling for phosphopeptide mapping by MALDI-MS and subsequent phosphopeptide sequencing by MALDI-MS/MS. The enhanced response of phosphopeptide ions in MALDI facilitates MS/MS of large (>3 kDa) multiply phosphorylated peptide species and reduces the amount of analyte needed for complete characterization of phosphoproteins.     

On-line affinity selection of histidine-containing peptides using a polymeric monolithic support for capillary electrophoresis

An on-line affinity selection method using a polymeric monolithic support is proposed for the retention of histidine-containing peptides and their subsequent separation by capillary zone electrophoresis (CZE). Monolithic capillary columns were prepared in fused-silica capillaries of 150 mum inner diameter (ID) by ionizing radiation-initiated in situ polymerization and cross-linking of diethylene glycol dimethacrylate and glycidyl methacrylate, and chemically modified with iminodiacetic acid (IDA) and copper ion. Monolithic microextractors were coupled on-line near the inlet of the separation capillary (fused-silica capillary, 75 mum ID x 28 cm from the microextractor to the detector). Model peptide mixtures of histidine-containing and histidine-noncontaining peptides were assessed. Peptides were released from the sorbent by a 5 mM imidazole solution and then separated by CZE with ultraviolet detection. Relative standard deviation values for migration times and corrected peak areas were found to be lower than 5.8 and 10.5%, respectively. IDA-Cu(II) ion modified monolithic microextractors showed a chromatographic behavior and could be reused at least 25 times. The use of monolithic supports proved to be an advantageous alternative to packed particles for the preparation of microextractors.     

Synthetic metal-binding protein surface domains for metal ion-dependent interaction chromatography. II. Immobilization of synthetic metal-binding peptides from metal ion transport proteins as model bioactive protein surface domains.

This preliminary investigation tests the premise that biologically relevant (1) peptide-metal ion interactions, and (2) metal ion-dependent macromolecular recognition events (e.g., peptide-peptide interactions) may be modeled by biomimetic affinity chromatography. Divinylsulfone-activated agarose (6%) was used to immobilize three different synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein histidine-rich glycoprotein (HRG). The synthetic peptides represented 1-3 multiple repeat units of the 5-residue sequence (Gly-His-His-Pro-His) found in the C-terminal of HRG. By frontal analyses, immobilized HRG peptides of the type (GHHPH)nG, where n = 1-3, were each found to have a similar binding capacity for both Cu(II) ions and Zn(II) ions (31-38 mumol/ml gel). The metal ion-dependent interaction of a variety of model peptides with each of the immobilized HRG peptide affinity columns demonstrated differences in selectivity despite the similar internal sequence homology and metal ion binding capacity. The immobilized 11-residue HRG peptide was loaded with Cu(II) ions and used to demonstrate selective adsorption and isolation of proteins from human plasma. These results suggest that immobilized metal-binding peptides selected from known solvent-exposed protein surface metal-binding domains may be useful model systems to evaluate the specificity of biologically relevant metal ion-dependent interaction and transfer events in vitro.     

Relative quantification of tau-related peptides using guanidino-labeling derivatization (GLaD) with online-LC on a hybrid ion trap (IT) time-of-flight (ToF) mass spectrometer

Development of a quantification method based on isotopic variants of O-methyl isourea (OMIU) in conjunction with reversed-phase (RP) liquid chromatography (LC) electrospray mass spectrometry is described for determining the relative quantification of tau-related peptides Ac-VQIVXK-NH2. Extracted ion chromatograms of the mass spectrometric data derived from online microcapillary LC separation identifies the retention times of the isotopically derivatized peptides together with their ion abundances. Data-dependent MSMS analysis of both derivatized variants of the same peptide provides a complementary method for identification and resolution between isobaric species. In addition, with respect to offline LC MALDI a larger number of analogues are detected and formation of amyloid is also observed for the aspartic acid and histidine-containing peptides.     

Multifunctional protein processing chip with integrated digestion, solid-phase extraction, separation and electrospray

We describe a microfluidic device in which integrated tryptic digestion, SPE, CE separation and electrospray ionization for MS are performed. The chip comprised of 10 × 30 μm channels for CE, and two serially connected 150?μm deep, 800?μm wide channels packed with 40 to 60 μm diameter beads, loaded with either immobilized trypsin, reversed-phase packing or both. On-chip digestion of cytochrome c using the trypsin bed showed complete consumption of the protein in 3 min, in contrast to the 2 h required for conventional solution phase tryptic digestion. SPE of 0.25 μg/mL solutions of the peptides leu-enkephalin, angiotensin II and LHRH gave concentration enhancements in the range of 4.4-12, for a ten times nominal volume ratio. A 100 nM cytochrome c sample concentrated 13.3 times on-chip gave a sequence coverage of 85.6%, with recovery values ranging from 41.2 to 106%. The same sample run without SPE showed only five fragment peaks and a sequence coverage of 41.3%. When both on-chip digestion and SPE (13.3 volume ratio concentration enhancement) were performed on 200 nM cytochrome c samples, a sequence coverage of 76.0% and recovery values of 21-105% were observed. Performing on-chip digestion alone on the same sample gave only one significant fragment peak. The above digestion/peptide concentration step was compared to on-chip protein concentration by SPE followed by on-chip digestion with solution phase trypsin. Both procedures gave similar recovery results; however, much larger trypsin autodigestion interference in the latter approach was apparent.     

Quantitation of human glutathione S-transferases in complex matrices by liquid chromatography/tandem mass spectrometry with signature peptides

Direct quantitation of glutathione S-transferase (GST) isoforms [alpha (GST-A) and micro (GST-M)] in human liver cytosol was achieved by liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) analysis of signature peptides of GST-A and GST-M and their corresponding stable isotopic peptide internal standards via multiple reaction monitoring (MRM). The selection of signature peptides was performed via trypsin digestion of commercially available cDNA-expressed GST-A1 and GST-M1, followed by LC/ESI-MS/MS with an ion trap mass spectrometer and sequencing with the TurboSEQUEST application. Quantitative analysis of the selected signature peptides in the multi-reaction monitoring (MRM) mode was performed using a triple-quadruple mass spectrometer. A series of human cytosol samples was quantitatively analyzed for levels of GST-A and GST-M. The total level of GST-A and GST-M obtained from this LC/ESI-MS/MS method was well correlated with the total level of GST determined by the 1-chloro-2,4-dinitrobenzene (CDNB) method.     

Metal-binding ability of human prion protein fragment peptides analyzed by column switch HPLC

The structural conversion of the prion protein (PrP) from the normal cellular isoform (PrP(C)) to the posttranslationally modified form (PrP(Sc)) is thought to relate to Cu2? binding to histidine (H) residues. Traditionally, the binding of metals to PrP has been investigated by monitoring the conformational conversion using circular dichroism (CD). In this study, the metal-binding ability of 21 synthetic peptides representing regions of human PrP(C) was investigated by column switch high-performance liquid chromatography (CS-HPLC). The CS-HPLC system is composed of a metal chelate affinity column and an octadecylsilica (ODS) reversed-phase column that together enable the identification of metal-binding regardless of conformational conversion. Synthetic peptides were designed with respect to the position of H residues as well as the secondary structure of human PrP (hPrP). The ability of the octapeptide (PHGGGWGQ)-repeating region (OP-repeat) to bind metals was analyzed by CS-HPLC and supported by CD analysis, and indicated that CS-HPLC is a reliable and useful method for measuring peptide metal-binding. Peptides from the middle region of hPrP showed a high affinity for Cu2?, but binding to Zn2?, Ni2?, and Co2? was dependent on peptide length. C-Terminal peptides had a lower affinity for Cu2?, Zn2?, Ni2?, and Co2? than OP-repeat region peptides. Interestingly, hPrP193-230, which contained no H residues, also bound to Cu2?, Zn2?, Ni2?, and Co2?, indicating that this region is a novel metal-binding site in the C-terminal region of PrP(C). The CS-HPLC method described in this study is useful and convenient for assessing metal-binding affinity and characterizing metal-binding peptides or proteins.     

磺化修饰结合离子交换色谱和生物质谱富集鉴定含组氨酸肽段

通过在肽段的N端引入磺酸基,从而使含组氨酸的肽段与其他肽段在pH<3.0的条件下产生电荷差异,建立了一种基于强阳离子交换色谱(SCX)结合生物质谱富集鉴定含组氨酸肽段的方法,并以含有组氨酸的标准蛋白质为模型,进行了方法学考察。结果表明,经N端磺酸化后,含组氨酸的肽段能有效地被阳离子交换色谱富集,且在肽的N端引入磺酸基促进了肽的裂解,使之产生简单而信息丰富的二级质谱图,从而得到完美的质谱鉴定结果。这说明磺化修饰结合强阳离子交换色谱用于含组氨酸肽段的富集鉴定是可行的,且具有在蛋白质组研究中应用的潜力。