Use of microbore high-performance liquid chromatography for purifying subnanomole levels of polypeptides for microsequencing. Structural studies on the murine plasma cell antigen PC-1

A procedure for the purification of subnanomole levels of polypeptides has been developed. Reversed-phase high performance liquid chromatography on short (10 cm or less) microbore (1-2 mm internal diameter) columns has been used to fractionate and purify a number of tryptic peptides generated from approximately 600 pmol of purified murine plasma cell antigen PC-1, a major membrane glycoprotein on all cells secreting immunoglobulins. The use of reversed-phase microbore columns permits the recovery of subnanomole amounts of polypeptides from large volumes in high yield (greater than 90%) and in small eluent volumes (40-60 microL) which can be loaded directly onto the gas-phase sequencer without further concentration. This procedure avoids the severe sample loss which frequently occurs with other concentration procedures such as lyophilization and evaporation. The use of a photodiode-array detector for identifying tryptophan-containing peptides from on-the-fly, ultraviolet spectra is described. This procedure permits the selection of tryptophan-containing peptides from complex tryptic digests for use as candidate peptides for oligonucleotide probe construction. Automated Edman degradation was performed on seven tryptic peptides, yielding 110 unique assignments; this corresponds to approximately 11% of the molecule.     

Peptide inhibitors of angiotensin I-converting enzyme in digests of gelatin by bacterial collagenase.

Peptide inhibitors of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) were produced by digesting gelatin with bacterial collagenase. The inhibitors were isolated from the digests with a combination of alcohol fractionation, treatment with Amberlite CG-50 column, gel filtration through Sephadex G-25, and Dowex 50 column and paper chromatography. Nine peptide fractions were purified to apparent homogeneity judging by thin-layer and ion-exchange column chromatography, and amino acid composition. Amino acid sequences of the peptides were determined: 2 were found to be mixtures of peptides and the sequence of another was only partially determined. Six of the peptides were potent inhibitors of the converting enzyme, while the other three were less active. 6 peptides were substrates for the enzyme. The enzyme released a dipeptide, Ala-Hyp from one peptide and was strongly inhibited by this dipeptide. The remainder of the parent peptides was a less effective inhibitor.     

The origin and control of ex vivo oxidative peptide modifications prior to mass spectrometry analysis

The origin and control of ex vivo sample handling related oxidative modifications of methionine-, S-alkyl cysteine-, and tryptophan-containing peptides obtained from typical "in-solution" or "in-gel" proteolytic digestion strategies, have been examined by capillary HPLC and MS/MS. The origin of increased oxidation levels were found to be predominantly associated with the extensive ex vivo sample handling steps required for gel electrophoresis and/or in-gel proteolytic digestion of proteins prior to analysis by MS. Conditions for deliberately controlling the oxidation state (both oxidation and reduction) of these peptides, as well as for those containing cysteine, have been evaluated using a series of model synthetic peptides and standard tryptic protein digests. Essentially complete oxidation of methionine- and S-alkyl cysteine-containing peptides was achieved by reaction with 30% hydrogen peroxide/5% acetic acid at room temperature for 30 min. Under these conditions, cysteine was also converted to cysteic acid, while only limited oxidation of tryptophan to oxindolylalanine, and methionine and S-alkyl cysteine sulfoxides to their respective sulfones, were observed. Efficient reduction of methionine- and S-alkyl cysteine sulfoxide-containing peptides was achieved by reaction in 1 M dimethylsulfide/10 M hydrochloric acid at room temperature for 10 and 45 min, respectively. None of the reduction conditions evaluated were found to result in the reduction of oxindolylalanine, cysteic acid, or methionine sulfone.     

Selective enrichment of tryptophan-containing peptides from protein digests employing a reversible derivatization with malondialdehyde and solid-phase capture on hydrazide beads

A method for the selective enrichment of tryptophan-containing peptides from complex peptide mixtures such as protein digests is presented. It is based on the reversible reaction of tryptophan with malondialdehyde and trapping of the derivatized Trp-peptides on hydrazide beads via the free aldehyde group of the modified peptides. The peptides are subsequently recovered in their native form by specific cleavage reactions for further (mass spectrometric) analysis. The method was optimized and evaluated using a tryptic digest of a mixture of 10 model proteins, demonstrating a significant reduction in sample complexity while still allowing the identification of all proteins. The applicability of the tryptophan-specific enrichment procedure to complex biological samples is demonstrated for a total yeast cell lysate. Analysis of the processed fraction by 1D-LC-MS/MS confirms the specificity of the enrichment procedure, as more than 85% of the peptides recovered from the enrichment step contained tryptophan. The reduction in sample complexity also resulted in the identification of additional proteins in comparison to the untreated lysate.     

Separation of peptides by reverse-phase high-performance liquid chromatography using propyl- and cyanopropylsilyl supports

The separation of peptides and proteins by reverse-phase high-performance liquid chromatography with cyanopropylsilyl and large-pore propylsilyl supports, together with aqueous trifluoroacetic acid/acetonitrile gradients, was studied. Operating parameters (trifluoroacetic acid concentration, flow rate, and gradient slope) were evaluated using different enzymatic digests of horse cytochrome c and bovine serum albumin. Peptides ranging in size from five amino acids to 68 kDa could be separated on the propylsilyl column in a single chromatographic run. The cyanopropylsilyl column is suitable as a supplement to the use of the large-pore column for medium size (5-20 amino acids) peptides. The chromatographic supports and conditions presented here offer a simple, sensitive, and rapid separation system for a wide size range of peptides and proteins. They extend the versatility of separation methodology for these molecules.     

Are the arginine kinase isoenzymes of Limulus polyphemus the product of two separate genes?

Limulus polyphemus has two electrophoretically distinct arginine kinases, a negative and a neutral (at pH 8.6). The highest concentrations of the neutral are found in muscle, where the total arginine kinase activity is highest. The peptide maps of the two forms are very similar, but the negative has six more acidic peptides than the neutral. Three tryptophan-containing peptides were isolated from tryptic digests of each form. The amino acid compositions of two of the three pairs of peptides were different. This sugests that the isoenzymes are the products of two distinct genes.Supported in part by American Cancer Society Grant #P-77L and by a grant from the National Cancer Institute of the National Institutes of Health #CA-11683-01.     

Purification and analysis of the major components of chum salmon protamine contained in insulin formulations using high-performance liquid chromatography

A simple high-performance liquid chromatographic method has been developed for the rapid purification and analysis of protamine components contained in insulin formulations. Only a single step is needed to separate peptides whose compositions, sizes, and unusual isoelectric points (pI 13.8) are nearly identical. The method involves their isocratic separation on a reversed-phase column using a pH 2 phosphate buffer and a low acetonitrile content as an eluant. The purified chum salmon components were analyzed by amino acid analysis, solid-phase amino acid sequencing, carboxypeptidase B digests, insulin complexation analysis, and a mass spectrophotometric procedure which gives an accurate mass of the intact peptides. This HPLC purification technique may also be applicable to protamines and other highly basic peptides isolated from other sources.     

Antigenic and calcium binding properties of a Peptide containing the essential cysteine in lima bean lectin

Polyclonal antisera were raised against a peptide containing the cysteine residue required for carbohydrate binding activity in the lima bean lectin. The antisera were tested for cross-reactivity with (a) synthetic peptide analogs to the essential cysteine containing peptide, (b) proteolytic digests of related lectins, (c) native lectins. The antisera were specifically inhibited from binding to a peptide conjugate by free synthetic peptides. The degree of inhibition by lectin digests correlated approximately along evolutionary relationships and the degree of sequence conservation. One antiserum was found to cross-react with certain lectins in the native state. In a second set of experiments, the calcium binding properties of the synthetic peptides were investigated using metal ion-chelate chromatography and UV-difference spectroscopy. The nonapeptide and undecapeptide bound to a Ca²⁺ iminodiacetic acid agarose column and were eluted with EDTA. Ultraviolet difference spectral titrations with Ca²⁺ performed on the synthetic undecapeptide and a related favin derived peptide resulted in dissociation constants of approximately 6 × 10³ per molar.     

Optimization of Immobilized Gallium (III) Ion Affinity Chromatography for Selective Binding and Recovery of Phosphopeptides from Protein Digests

Although widely used in proteomics research for the selective enrichment of phosphopeptides from protein digests, immobilized metal-ion affinity chromatography (IMAC) often suffers from low specificity and differential recovery of peptides carrying different numbers of phosphate groups. By systematically evaluating and optimizing different loading, washing, and elution conditions, we have developed an efficient and highly selective procedure for the enrichment of phosphopeptides using a commercially available gallium(III)-IMAC column (PhosphoProfile, Sigma). Phosphopeptide enrichment using the reagents supplied with the column is incomplete and biased toward the recovery and/or detection of smaller, singly phosphorylated peptides. In contrast, elution with base (0.4 M ammonium hydroxide) gives efficient and balanced recovery of both singly and multiply phosphorylated peptides, while loading peptides in a strong acidic solution (1% trifluoracetic acid) further increases selectivity toward phosphopeptides, with minimal carryover of nonphosphorylated peptides. 2,5-Dihydroxybenzoic acid, a matrix commonly used when analyzing phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry was also evaluated as an additive in loading and eluting solvents. Elution with 50% acetonitrile containing 20 mg/mL dihydroxybenzoic acid and 1% phosphoric acid gave results similar to those obtained using ammonium hydroxide as the eluent, although the latter showed the highest specificity for phosphorylated peptides.     

Obtaining high sequence coverage in matrix-assisted laser desorption time-of-flight mass spectrometry for studies of protein modification: analysis of human serum albumin as a model

Several approaches were explored for obtaining high sequence coverage in protein modification studies performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Human serum albumin (HSA, 66.5kDa) was used as a model protein for this work. Experimental factors considered in this study included the type of matrix used for MALDI-TOF MS, the protein digestion method, and the use of fractionation for peptide digests prior to MALDI-TOF MS analysis. A mixture of alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid was employed as the final matrix for HSA. When used with a tryptic digest, this gave unique information on only half of the peptides in the primary structure of HSA. However, the combined use of three enzyme digests based on trypsin, endoproteinase Lys-C, and endoproteinase Glu-C increased this sequence coverage to 72.8%. The use of a ZipTip column to fractionate peptides in these digests prior to analysis increased the sequence coverage to 97.4%. These conditions made it possible to examine unique peptides from nearly all of the structure of HSA and to identify specific modifications to this protein (e.g., glycation sites). For instance, Lys199 was confirmed as a glycation site on normal HSA, whereas Lys536 and Lys389 were identified as additional modification sites on minimally glycated HSA.     

The amino acid sequence around four cysteine residues in trout actin

Four unique carboxymethylcysteine-containing peptides were isolated from tryptic and chymotryptic digests of trout muscle actin carboxymethylated with iodo[2-(14)C]acetic acid in 6m-guanidinium chloride. The amino acid sequences of these peptides were determined and showed a high degree of homology with the corresponding sequences from rabbit actin. One of the radioactive peptides was the C-terminal peptide and another sequence probably contained the cysteine residue from the N-terminal region of the protein.     

Facile analysis and purification of deblocked N-terminal pyroglutamyl peptides with a strong cation-exchange sulfoethyl aspartamide column

A high-performance strong cation-exchange Sulfoethyl Aspartamide column was used to analyze and purify five N-terminal pyroglutamyl peptides after treatment with Pyroglutamate Aminopeptidase. The resulting deblocked N-1 peptides possess an increased positive charge and are therefore retained to a greater extent by the column. Salt gradient elution in a pH 3 mobile phase was then used to recover the desired peptides and the purified deblocked peptides were directly subjected to N-terminal sequence analysis. The same digests were also chromatographed on a C18 reversed-phase column using standard trifluoroacetic acid-acetonitrile gradient elution. The elution order for the parent peptide and the N-1 peptide on the reversed-phase column was reversed from that on the Sulfoethyl Aspartamide column and the resolution of the two peptides obtained on the reversed-phase column was less than that observed on the cation-exchange column. In addition, the Sulfoethyl Aspartamide column was shown to be useful to monitor the extent of N-terminal glutamine cyclization formed during peptide purification and storage.     

Immobilized anhydrotrypsin as a biospecific affinity adsorbent for the peptides produced by trypsin-like proteases.

Various naturally occurring peptides containing l-arginine at the carboxyl termini were tightly adsorbed at pH 5 on anhydrotrypsin, a chemical derivative of bovine trypsin, immobilized on Sepharose, and desorbed by washing with 5 mm HCl. The largest of the peptides examined was Fragment 2 (“histidine-rich peptide”) with 41 amino acid residues, which had been released from bovine high-molecular-weight kininogen by plasma kallikrein. When only the carboxyl-terminal arginine was removed by carboxypeptidase B, the peptides lost their specific affinity toward the immobilized anhydrotrypsin. The peptide fragments in the tryptic digests of reduced and S-carboxymethylated erabutoxin a were also fractionated effectively by chromatography on this affinity adsorbent. The fragments containing l-lysine at the carboxyl termini showed weaker affinity for the adsorbent than those containing l-arginine at the termini.     

THE ISOLATION AND PROPERTIES OF LARGE BASIC PEPTIDES FROM BOVINE SPINAL CORD

Abstract— Two basic peptides (B1 and B2) were derived from bovine spinal cord following in situ proteolysis at 37°C for 10–24 h. These peptides do not arise as degradation products from the A1 protein as shown by amino acid composition and end group analysis; rather they appear to originate from some larger basic protein in the spinal cord having similarities to the P2 protein, a basic protein found in peripheral nerve myelin. The peptides were purified following defatting, acid extraction, and ammonium sulphate fractionation, by chromatography on Amberlite IRC-50 resin using guanidinium chloride. The peptides, found generally in a 4:1 ratio of B1 to B2, appeared homogeneous on gel electrophoresis and immunodiffusion. Approximately 25–60 mg of peptides was obtained per 100 g wet spinal cord.
In contrast to the basic A1 protein from myelin, neither of these peptides nor their pepsin digests were encephalitogenic. They do not cross-react immunologically with the basic A1 protein, but cross-react with each other. These peptides further differ from the A1 protein in their tryptic peptide map, size (B1, 63 residues; B2, 54 residues), and composition particularly the high lysine: arginine ratio, and low histidine content. Like the A1 protein, however, they contain a tryptophan residue and a blocked NH₂-terminal amino acid; peptide Bl has COOH-terminal valine. It was concluded that the basic peptides represent a fragment of a hitherto unidentified protein(s) of the nervous system.     

Primary structure of 20S,22R-cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria. IV. Structure of peptides of thermolytic and limited tryptic hydrolysis of the fragment F1; peptides of cyanogen bromide hydrolysis of cytochrome P-450. Complete amino acid sequence

A thermolytic hydrolysis of maleinated fragment F1 has been performed, resulted in isolation of 44 peptides; their complete amino acid sequence has been determined. Non-overlapping thermolytic peptides of fragment F1 involve 178 amino acid residues, which comprises about 71% of its amino acid sequence. Also, the cleavage and structural investigation of some tryptophan-containing peptides obtained from the limited trypsinolysis of fragment F1 were carried out; reconstitution of the polypeptide chain of the fragment is completed. The cyanogen bromide cleavage of carboxymethylated cytochrome P-450 was achieved and 17 peptides, comprising almost the whole polypeptide chain of the protein molecule (91%), was isolated. To investigate structure of the cyanogen bromide peptides, we hydrolysed them at tryptophan residues with trypsin, chymotrypsin, proteinase from Staphylococcus aureus, and BNPS-skatole. The data obtained and those published earlier led to the complete primary structure of the cholesterol-hydroxylating cytochrome P-450. The proteins polypeptide chain consists of 481 amino acid residues and has the precise molecular mass 56 407.7.     

A diagonal electrophoretic method for selective purification of methionine peptides

1. A method is described that selectively purifies methionine peptides from enzymic digests of a protein. The peptides, after paper electrophoresis, are treated on paper with iodoacetamide at acid pH. This specifically converts methionine residues into their sulphonium salts. When the paper is submitted to electrophoresis at right angles to the original direction, the carbamoylmethylmethionine peptides emerge from an undifferentiated diagonal. 2. Heating at neutral pH converts carbamoylmethylmethionine into homoserine and thereby specifically cleaves the peptides. 3. The effect of the modifications on amino acid composition and sequence analyses of the peptides was studied. 4. When the method was applied to a tryptic digest of S-aminoethyl-chymotrypsinogen A, two peptides were selectively purified that had the expected amino acid sequence.     

N-terminal sequence of soybean beta-amylase

The blocked N-terminus and N-terminal sequence of soybean beta-amylase were determined by analyzing the acidic peptides derived on peptic digestion of the enzyme. The acidic peptides were separated from the digest on a Dowex 50 X 2 column and purified by reversed phase-high performance liquid chromatography (RP-HPLC). The major acidic peptide, Pep-4, was a heptapeptide with a molecular weight of 766. Forty-eight hundredths mol acetyl group and 0.61 mol acetyl-Ala per mol of Pep-4 were detected on RP-HPLC analysis. The N-terminal 9 amino acid sequence of soybean beta-amylase was deduced to be acetyl-Ala-Thr-Ser-Asp-Ser-Asn-Met-(Gly-Leu) from the results of sequence analysis of Pep-4 and amino acid analysis of other acidic peptides.     

A method for the identification of glycoproteins from human serum by a combination of lectin affinity chromatography along with anion exchange and Cu-IMAC selection of tryptic peptides

This paper reports a method for identifying glycoproteins from human serum. Glycoproteins were selected with a concanavalin A (Con A) lectin column and then tryptically digested prior to sequential chromatographic selection of acidic and histidine containing peptides. Acidic peptides were selected with a strong anion exchange (SAX) column. Peptides captured by the SAX columns were then released and histidine-containing peptides in the mixture selected with a copper loaded immobilized metal affinity chromatography (Cu-IMAC) column. This serial chromatographic selection process reduced the complexity of proteolytic digests by more than an order of magnitude. Peptides selected by this serial process were then fractionated by reversed-phase chromatography (RPC) and identified by tandem mass spectrometry. The method was initially validated using human transferrin before application to human serum. The results show that all the peptides identified except one contained histidine and acidic amino acids.     

Amino acid sequence of the beta subunit of follicle-stimulating hormone from human pituitary glands.

The beta subunit of follicle-stimulating hormone (FSH-beta) from human pituitary glands was reduced and S-aminoethylated prior to thermolytic, tryptic, and chymotryptic digestions. Each digest was gel-filtered on Sephadex G-50 to seperate the glycopeptides. The glycopeptides and the peptides were isolated by high voltage paper electrophoresis at pH 6, 3.5, and 2.0. The purity of the isolated peptides was confirmed by amino acid analyses. The amino acid sequences of peptides were determined by Edman degradation followed by subtractive amino acid analysis and, in certain cases, confirmed by dansylation. COOH-terminal sequences of the peptides were determined by digestion with carboxypeptidases A and B and by hydrazinolysis. The tryptophan content of human follicle-stimulating hormone, of the beta subunit of human follicle-stimulating hormone, and of the glycopeptides obtained from the enzymic digests was determined by fluorescence spectra, titration against N-bromosuccinimide, colorimetric estimation with p-dimethyl aminobenzaldehyde, hydrolysis with methane sulfonic acid containing 0.2% tryptamine followed by amino acid analysis, microbiological assay, and sequence analysis. The presence of 1 tryptophan residue in the beta subunit was indicated.     

The occurrence of extended acidic sequences in nonhistone chromosomal proteins

Nonhistone chromosomal proteins and soluble cytoplasmic proteins from rat liver were treated with a combination of proteases and chemical reagents which split a variety of peptide bonds but do not attack sequences consisting predominantly or exclusively of acidic amino acid residues. Analysis of the resulting digests by gel filtration chromatography and column electrophoresis demonstrated that, relative to cytoplasmic proteins, nonhistone chromosomal proteins are rich in highly charged, acidic peptides up to 12 residues in length, but rarely contain very long peptides consisting exclusively of acidic residues such as are found in the nonhistone chromosomal proteins HMG1 and HMG2.