Microbore HPLC/mass spectrometry for the analysis of peptide mixtures using a continuous flow interface

Microbore HPLC techniques have been combined with fast atom bombardment mass spectrometry to provide HPLC/MS capabilities for the analysis of mixtures of peptides and small proteins. The interface between the liquid chromatograph and mass spectrometer is a continuous flow direct insertion probe which contains a fused silica capillary that delivers the eluting solvent to the FAB source of the mass spectrometer at a rate of 5-10 microL/min. Data are presented for the analysis of several mixtures of peptides ranging in molecular weights from about 900 to 6000 daltons. In addition, the analysis of 100 pmol of a tryptic digest of whale myoglobin is shown where 16 of the possible 19 peptides were identified in the mass range m/z 2200-250. The advantages of this approach to HPLC/MS are a relatively high sensitivity because of the low flow rates and low background, and the ability to detect high molecular weight compounds.     

Peptide synthesis in aqueous-organic solvent mixtures with alpha-chymotrypsin immobilized to tresyl chloride-activated agarose

alpha-Chymotrypsin was immobilized with a high coupling yield (up to 80%) to tresyl chloride activated Sepharose CL-4B.The immobilized enzyme was tested for its ability to synthesize soluble peptides from N-acetylated amino acid esters as acyl donors and amino acid amides as acceptor amines in water-water-miscible organic solvent mixtures. It was found that the yield of peptide increased with increasing concentration of organic cosolvent. Almost complete synthesis (97%) of Ac-Phe-Ala-NH(2) was obtained from Ac-Phe-OMe using a sixfold excess of Ala-NH(2). The rate of peptide formation in aqueous-organic solvent mixtures was good. Thus, 0.1M peptide was formed in less than 2 h in 50 vol% DMF with 0.1 mg immobilized chymotrypsin/mL reaction mixture. The immobilized enzyme distinguished between the L and D configurations of acceptor amino acid amides even in high concentration of nonaqueous component (90% 1,4-butanediol). The effect of temperature was studied. It was found that both the yield of peptide and the stability of immobilized enzyme increased when the temperature was lowered. Experiments could be performed at subzero temperatures in the aqueous-organic solvent mixtures resulting in very high yield of peptide. After three weeks continuous operation at 4 degrees C in 50% DMF, the immobilized enzyme retained 66%of its original synthetic activity. The activity of the immobilized enzyme was better conserved with a preparation made from agarose with a higher tresyl group content compared to a preparation made from a lower activated agarose, indicating that multiple point of attachment has a favorable effect on the stability of the enzyme in aqueous-organic solvent mixtures. The major advantage of using water-miscible instead of water-immiscible organic solvents to promote peptide syntheses appears to be the increased solubility of substrates and products, making continuous operation possible.     

Correlation of secondary structure with biological activity for a leader peptide: circular dichroism-derived structure and in vitro biological activities of preproparathyroid hormone peptide and its analogs

Leader or signal sequences are specialized domains within precursor proteins which serve an essential role in interacting with the cellular secretory apparatus to enable intracellular transport and secretion of proteins. Despite many differences in primary amino acid sequences, signal domains interact with a common set of intracellular components, presumably because the signal sequences share an overall conformational similarity. In a few instances, mutant signal peptides from prokaryotes have been studied and their structures correlated with function (export) in vivo. A series of analogs of the precursor-specific region of preproparathyroid hormone have been prepared which contain substitutions of either proline or a charged amino acid within the hydrophobic core. These synthetic "mutants" have previously been evaluated in several in vitro assays to determine their functionality with regard to protein secretion and suitability as substrates for signal peptidase. The secondary structural content of each peptide, as well as the native sequence and sulfur-free analog, was determined in aqueous and nonaqueous conditions by circular dichroism (CD) as a function of time. The structures obtained were correlated with in vitro bioactivities. Unlike the findings or previous CD studies, all the peptides examined here had low to undetectable alpha-helical content in both aqueous and nonaqueous buffers. The unsubstituted and sulfur-free analogs had high (80-85%) beta-structure in aqueous conditions which was reduced to approximately 30% in nonaqueous solvent. The proline- and charged-substituted peptides contained about half the beta-structure content (35-55%) in aqueous buffer; in nonaqueous solvent their structure was similar to the unsubstituted peptides. The structure-activity correlates found were as follows: a high degree of structure (aqueous conditions) correlated with interaction with signal recognition particle and substrate suitability for signal peptidase; a low degree of structure (nonaqueous environment) correlated with activity in the translocation assay.     

Optimization of the binding properties of a synthetic anion receptor using rational and combinatorial strategies

The solution conformations of tetrameric and hexameric cyclopeptides containing alternating L-proline and 6-aminopicolinic acid subunits strongly depend on solvent polarity. Whereas in polar solvents, such as d6-DMSO, both peptides prefer on average symmetric conformations with converging NH groups, in less polar chloroform intramolecular hydrogen bonds to the peptide NH groups stabilize other, and in the case of the hexapeptide, non-symmetrical conformations. Independent of the solvent, both peptides interact with anions via their NH groups but whereas anion binding requires a cleavage of the intramolecular hydrogen bonds accompanied by a conformational reorganization in chloroform, in polar solvents the peptides are already well preorganized for anion complexation. Complex formation between anions and the cyclic hexapeptide was even detected in highly competitive D2O/CD3OD or H2O/CH3CN mixtures, which was attributed to the special sandwich-type structure of the complexes formed. Stabilizing these 2:1 aggregates by covalently linking two cyclopeptide rings together affords ditopic receptors with a high anion affinity in protic solvents. Complex stability depends on the structure of the linker with which the two receptor moieties are connected and even more potent anion receptors were obtained by a dynamic combinatorial optimization of this linking unit.     

Capillary electrophoretic separation of proteins and peptides by ion-pairing with heptanesulfonic acid

Heptanesulfonic acid as ion-pairing agent was used for the separation of mixtures of low and high molecular mass peptides/proteins by capillary electrophoresis. The separation conditions used were: capillary 37 cm (30 cm to the detector) x 75 microm i.d., voltage 10 kV, phosphate buffer 50 mmol/l, ion-pairing agent heptanesulfonic acid at three different concentrations, namely, 0, 20 or 100 mmol/l, pH 2.5. The separation reflected the ion-pairing equilibria between the ion-pairing agent and the peptide/protein analytes. The influence of ion-pairing on sample mobility (running time) was more pronounced in case of the higher-molecular peptides as compared to the low molecular ones. This difference offers the possibility to separate low and high molecular peptides/proteins that under the absence of the ion-pairing agent would co-migrate. The principle of this approach was demonstrated on a randomly selected set of peptides/proteins; the practical applicability was demonstrated on a set of CNBr peptides arising from a naturally occurring mixture of collagen types I and III.     

Combinations of turns in proteins.

We observed that beta- and gamma-turns in protein structure may be associated as peptides representing combinations of turns that span between nine and 26 amino acid residues along the polypeptide backbone chain and often correspond to loops in the protein structure. Around 475 peptides resulted from the analysis of a non-redundant data set corresponding to 248 protein crystal structures selected from the Protein Data Bank. Nearly 40% protein chains are associated with two or more peptides and the peptides with nine and 10 amino acid residues are more frequent. A maximum of four distinct peptides varying in number of amino acid residues were observed in at least 10 proteins along the same protein chain. Nearly 80% peptides comprise type IV beta-turns that are associated with irregular dihedral angle values suggesting this may be important for the conformational diversity associated with the loops in proteins. In general, predominant interactions that possibly stabilize these peptides involve main-chain and side-chain interactions with solvent, in addition to hydrogen bond, salt-bridge and non-bonded interactions. Majority of the peptides were observed in hydrolase, oxidoreductase, transferase, serine proteinase/inhibitor complex, electron transport/electron transfer and lyase proteins.     

The structure and amount of tubulin in cells and tissues.

The NH2-terminals and amino acid compositions of three carboxymethylcysteine-containing peptides produced from carboxymethylated calf brain tubulin by digestion with elastase have been determined. The 3H/14C ratio obtained when these peptides were generated from mixtures of [14C]carboxymethylated calf brain tubulin and the [3H]carboxymethylated proteins of mouse brain or 3T3 cells and subsequently purified indicated that both mouse brain and 3T3 cells contained protein giving rise to peptides homologous to these peptides. This indicates that the tubulins of calf brain, mouse brain, and 3T3 cells are homologous in the regions of amino acid sequence accounting for the peptides examined. From the 3H/14C ratio of the isolated peptides, 13.5% of the protein of mouse brain and 3.5% of the protein of 3T3 cells were estimated to be tubulin.     

Reducer driven baric denaturation and oligomerisation of whey proteins

The influence of high pressure on alpha-lactalbumin (ALA)/beta-lactoglobulin (BLG) mixtures of various compositions was studied at pH 8.5 by gel-permeation chromatography and sodium dodecyl sulphate (SDS) gel electrophoresis without 2-mercaptoethanol. High-molecular protein disulfide oligomers formed after denaturation by the pressure of 10 kbar (1000 MPa) if the weight fraction of BLG (W(BLG)(0)) in the protein mixture exceeded 0.2. The maximum yield of these oligomers of order 80-85% is observed at W(BLG)(0)>/=0.4. Conversions of both proteins in the oligomers are roughly the same. The estimates of the oligomerisation yield obtained by the gel-permeation chromatography and SDS gel electrophoresis agree well. This indicates that the formation of intermolecular disulfide bonds is necessary for the oligomerisation. Thus, the oligomerisation of pressure denatured ALA and BLG is driven by the thiol<-->disulfide exchange rendered possible by the vigorous baric denaturation and the exposure of the free thiol group of BLG, which acts as an initiator of disulfide bridges scrambling.     

Extraction method for analysis of detergent-solubilized bacteriorhodopsin and hydrophobic peptides by electrospray ionization mass spectrometry

The analysis of integral membrane proteins or transmembrane peptides by electrospray ionization mass spectrometry (ESI-MS) is difficult since detergents, used to solubilize these hydrophobic proteins and peptides, severely suppress analyte ion formation. This problem has been addressed previously by precipitating the protein, removing the detergent, and resolubilizing the protein in a nonpolar solvent. Here, we demonstrate a method that avoids protein precipitation and resolubilization. Detergent-solubilized bacteriorhodopsin is extracted into a nonpolar solvent phase by adding a chloroform/methanol/water solvent mixture to the aqueous detergent solution. ESI mass spectra of the nonpolar, chloroform-rich phase were dominated by peaks due to bacterioopsin. Bacterioopsin precursors with partially cleaved leader sequences were seen in all mass spectra. Additional peaks were likely due to intact bacteriorhodopsin, i.e., bacterioopsin with the retinal prosthetic group attached, and to bacterioopsin associated with lipid molecules. A separation process that occurred in the fused-silica capillary leading to the electrospray tip was essential for obtaining ESI mass spectra of bacterioopsin. The extraction-into-chloroform procedure also worked well with hydrophobic, transmembrane-type peptides that were insoluble in other electrospray solvents, including 100% formic acid, and the method has application to transmembrane peptides formed from digests of integral membrane proteins.     

Design of synthetic gene libraries encoding random sequence proteins with desired ensemble characteristics.

Libraries of random sequence polypeptides are useful as sources of unevolved proteins, novel ligands, and potential lead compounds for the development of vaccines and therapeutics. The expression of small random peptides has been achieved previously using DNA synthesized with equimolar mixtures of nucleotides. For many potential uses of random polypeptide libraries, concerns such as avoiding termination codons and matching target amino acid compositions make more complex designs necessary. In this study, three mixtures of nucleotides, corresponding to the three positions in the codon, were designed such that semirandom DNA synthesized by repeated cycles of the three mixtures created an open reading frame encoding random sequence polypeptides with desired ensemble characteristics. Two methods were used to design the nucleotide mixtures: the manual use of a spreadsheet and a refining grid search algorithm. Using design targets of less than or equal to 1% stop codons and an amino acid composition based on the average ratios observed in natural, globular proteins, the search methods yielded similar nucleotide ratios, Semirandom DNA, synthesized with a designed, three-residue repeat pattern, can encode libraries of very high diversity and represents an important tool for the construction of random polypeptide libraries.     

Comparison of molecular dynamics simulation methods for amyloid β(1-42) monomers containing D-aspartic acid residues for predicting retention times in chromatography

Molecular dynamics simulations of amyloid β(1-42) containing D-aspartic acid residues were performed using several continuous solvent models to investigate the usefulness of simulation methods for D-amino acid-containing proteins and peptides. Normal molecular dynamics simulations and replica exchange molecular dynamics simulations, which are one of the generalized-ensemble algorithms, were performed. Because the β-structure contents of amyloid β(1-42) peptides obtained by replica exchange molecular dynamics simulations with Onufriev-Bashford-Case generalized Born implicit solvent were qualitatively consistent with experimental data, replica exchange molecular dynamics rather than other methods appeared to be more reasonable for calculations of amyloid β(1-42) containing D-aspartic acid residues. Computational results revealed that peptides with stereoinversion of Asp23 tend to form β-sheet structures by themselves, in contrast to the wild-type peptides that form β-sheet structures only after aggregation. These results are expected to be useful for computational investigations of proteins and peptides such as prediction of retention time of peptides and proteins containing D-aspartic acid residues.     

A high-yield method for the isolation of hydrophobic proteins and peptides from polyacrylamide gels for protein sequencing

A methodological approach is described which allows the isolation of hydrophobic and hydrophilic proteins and peptides in high yield. The technique consists of (1) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) protein elution from polyacrylamide gels with an organic solvent mixture composed of formic acid/acetonitrile/isopropanol/H2O (50/25/15/10, v/v/v/v), and (3) purification of eluted proteins by size exclusion chromatography on a Superose 12 column using this organic solvent mixture as eluant. The efficiency of this technique was tested with radioactively labeled polypeptides. These proteins were reaction center from Chloroflexus aurantiacus, bacteriorhodopsin, halorhodopsin from Halobacterium halobium, bovine serum albumin, ovalbumin, alpha-chymotrypsinogen A, and cytochrome c. The elution recoveries from polyacrylamide gels were 77-95%; the final yield after chromatographic purification was still 67-76% (with one exception). Subsequent amino acid sequencing was possible without further sample treatment. The sensitivity of the method described was found to be at least 20-30 micrograms protein.     

Reverse-phase high-performance liquid chromatography of hydrophobic proteins and fragments thereof

Reverse-phase high-performance liquid chromatography (HPLC) resolution and recovery of cytochrome P-450 and bovine rhodopsin, both integral membrane proteins, and large peptides derived from P-450 LM₂ were enhanced by utilizing ternary solvents. Surprisingly, most test materials eluted later in the gradient when using mixtures of acetonitrile and propanol in the mobile phase compared to using either solvent alone. Of the supports tested, the best recovery of hydrophobic cytochrome P-450 LM₄ was experienced on the less retentive CN-bonded phase. Two alternate solvents for HPLC of polypeptides are proposed: (1) 0.02–0.1 m hexafluoroacetone/NH₃, pH 7.2 for highly acidic peptides; and (2) 6 m formic acid/0.13 m trimethylamine, pH 1.5, vs 4 m formic acid/0.09 m trimethylamine in propanol for relatively insoluble peptides. Anomalous side reactions between formic acid and peptides can cause HPLC peak broadening, increased retention, and decreased resolution. These deleterious effects are thought to be due in part to formyl esterification of serine and threonine residues and appear to be reversible by aminoethanol treatment.     

Measurement of preferential solvation of some glucans in mixed solvent systems by gel-permeation chromatography

Preferential solvation of the glucans amylose, pullulan, and dextran in binary dimethyl-sulfoxide/water (DMSO/H₂O) solvent mixtures has been measured using gel-permeation chromatography. The preferential solvation behavior of the three glucans in DMSO/H₂O solvent mixtures is indistinguishable in the experiments reported. In solvent mixtures with mol ratio DMSO/H₂O less than 1:2, all three glucans are solvated preferentially by H₂O. The maximum extent of preferential solvation by H₂O is about 2.5 mol H₂O/mol of glucose residues. When the DMSO/H₂O mol ratio exceeds 1:2, DMSO solvates the glucans preferentially to a maximum extent of about 1 mol DMSO/mol of glucose residues. An interpretation of the change in preferential solvation with mixed solvent composition is suggested in terms of the known characteristics of the binary solvent system, and the relationship of preferential solvation, reported here, to the absolute solvation of the glucan chains is discussed.     

Investigation of proteins and peptides from yeastolate and subsequent impurity testing of drug product

Hydrolysates play an important role in modern biological production. These mixtures are mostly undefined and contain a mixture of proteins, peptides, and amino acids along with other non–amino acid‐based components. Recently, there has been an interest in defining and sequencing proteins and peptides in these hydrolysates to subsequently develop an assay to ensure removal during product purification. This work investigates an ultrafiltrate of yeastolate to determine whether any protein is present. Size exclusion chromatography indicated a possible high molecular weight component (>10 kDa). This suspected high molecular weight fraction was collected and investigated. It was determined that this fraction consists of nucleic acids; and no protein was detected using sensitive modern techniques including HPLC, mass spectrometry, and SDS‐PAGE. Next, five unique, yeast‐specific peptides were identified, sequenced, and confirmed. Finally, an impurity assay for any residual yeast specific peptides was developed and the analytical metrics were determined including accuracy, precision, linearity, range, and limits of detection and quantitation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Structure and solvation of melittin in 1,1,1,3,3,3-hexafluoro-2-propanol/water

Fluorinated alcohols can induce peptides and proteins to take up helical conformations. Nuclear Overhauser effect (NOE) spectroscopy experiments and analysis of C(alpha)H proton chemical shifts show that the conformation of melittin in 35% hexafluoro-2-propanol/water is alpha-helical from residues Ile-2 to Val-8 and from Leu-13 to Gln-25. As has been found in other solvent systems, the two helical regions are not colinear; the interhelix angle (73 +/- 15 degrees ) in 35% 1,1,1,3,3,3-hexafluoro-2-propanol/water is smaller than the angle found in other fluoroalcohol-water mixtures or in the crystal. Intermolecular (1)H(19)F and (1)H(1)H nuclear Overhauser effects were used to explore interaction of solvent components with melittin dissolved in this solvent mixture. The NOEs observed indicate that fluoroalcohol and water molecules are both tightly bound to the peptide in the vicinity of the interhelix bend. For the remainder of the molecule, solute-solvent NOEs are consistent with preferential solvation of the peptide by the fluoroalcohol component of the solvent mixture.     

Assignment of disulfide bonds in proteins by partial acid hydrolysis and mass spectrometry

A new method is described for locating disulfide bonds in proteins which cannot be cleaved between half-cystinyl residues by enzymic methods, as is often the case for tightly coiled proteins, or for proteins in which half-cystinyl residues are not separated by residues required for enzymic cleavage. Partial acid hydrolysis of a model protein, hen egg-white lysozyme, produces a mixture of disulfide-containing peptides from which the disulfide connections may be deduced. The usefulness of a combination of HPLC, fast atom bombardment mass spectrometry, and computer-assisted analysis to identify disulfide-containing peptides present in the partial acid hydrolysate of the model protein is demonstrated. Chromatographic fractions of the hydrolysate were analyzed by mass spectrometry before and after chemical reduction of the disulfide bonds to determine the molecular weights of disulfide-containing peptides. Computer-assisted analysis was then used to relate the molecular weights of these peptides to specific segments of the protein from which the disulfide connectivities could be determined. Partial acid hydrolysis of proteins, which is attractive because it proceeds relatively independent of the amino acid sequence and structure, and because disulfide interchange is unlikely to occur in dilute acid, has become practical because disulfide-containing peptides present in complex mixtures can be identified rapidly and definitively by this method.     

Purification of the c-erbB2/neu membrane-spanning segment:: a hydrophobic challenge

High quality purification of membrane-spanning peptides and proteins remains a challenging problem. In this work we describe a tailored chromatographic purification of a synthetic 35-residue peptide corresponding to the transmembrane region of the tyrosine kinase receptor c-erb2/neu. Composed to over 70% by the amino acids alanine, isoleucine, leucine, phenylalanine and valine, this peptide presents a very hydrophobic character. Product isolation from the complex peptide mixture, obtained after acid cleavage of the resin matrix used during the solid-phase synthesis, represents a difficult task. We propose a three step strategy based on gel permeation and reversed-phase high-performance liquid chromatography, using aprotic polar solvent mixtures. The challenge consisted in obtaining a sufficient amount of an extremely pure sample, in order to allow structural analysis by NMR spectroscopy. Keeping trace of the synthetic peptide throughout the different purification steps was assured by MALDI TOF mass spectrometry, and the final product purity was checked by coupled liquid chromatography–ESI TOF mass spectrometry.     

A metal-coded affinity tag approach to quantitative proteomics

The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures.     

Definition and characterization of a "trypsinosome" from specific peptide characteristics by nano-HPLC-MS/MS and in silico analysis of complex protein mixtures

Although HPLC-ESI-MS/MS is rapidly becoming an indispensable tool for the analysis of peptides in complex mixtures, the sequence coverage it affords is often quite poor. Low protein expression resulting in peptide signal intensities that fall below the limit of detection of the MS system in combination with differences in peptide ionization efficiency plays a significant role in this. A second important factor stems from differences in physicochemical properties of each peptide and how these properties relate to chromatographic retention and ultimate detection. To identify and understand those properties, we compared data from experimentally identified peptides with data from peptides predicted by in silico digest of all corresponding proteins in the experimental set. Three different complex protein mixtures extracted were used to define a training set to evaluate the amino acid retention coefficients based on linear regression analysis. The retention coefficients were also compared with other previous hydrophobic and retention scale. From this, we have constructed an empirical model that can be readily used to predict peptides that are likely to be observed on our HPLC-ESI-MS/MS system based on their physicochemical properties. Finally, we demonstrated that in silico prediction of peptides and their retention coefficients can be used to generate an inclusion list for a targeted mass spectrometric identification of low abundance proteins in complex protein samples. This approach is based on experimentally derived data to calibrate the method and therefore may theoretically be applied to any HPLC-MS/MS system on which data are being generated.