The ABRF Edman Sequencing Research Group 2008 Study: investigation into homopolymeric amino acid N-terminal sequence tags and their effects on automated Edman degradation.

The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis.

ABRF ESRG 2006 study: Edman sequencing as a method for polypeptide quantitation.

The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for protein and peptide analysis. These studies provide a means for participating laboratories to compare their analyses against a benchmark of those from other laboratories that provide this valuable service. The main purpose of the 2006 study was to determine how accurate Edman sequencing is for quantitative analysis of polypeptides. Secondarily, participants were asked to identify a modified amino acid residue, N-epsilon-acetyl lysine [Lys(Ac)], present within one of the peptides. The ESRG 2006 peptide mixture consisted of three synthetic peptides. The Peptide Standards Research Group (PSRG) provided two peptides, with the following sequences: KAQYARSVLLEKDAEPDILELATGYR (peptide B), and RQAKVLLYSGR (peptide C). The third peptide, peptide C*, synthesized and characterized by ESRG, was identical to peptide C but with acetyl lysine in position 4. The mixture consisted of 20% peptide B and 40% each of peptide C and its acetylated form, peptide C*. Participating laboratories were provided with two tubes, each containing 100 picomoles of the peptide mixture (as determined by quantitative amino acid analysis) and were asked to provide amino acid assignments, peak areas, retention times at each cycle, as well as initial and repetitive yield estimates for each peptide in the mixture. Details about instruments and parameters used in the analysis were also collected. Participants in the study with access to a mass spectrometer (MALDI-TOF or ESI) were asked to provide information about the relative peak areas of the peptides in the mixture as a comparison with the peptide quantitation results from Edman sequencing. Positive amino acid assignments were 88% correct for peptide C and 93% correct for peptide B. The absolute initial sequencing yields were an average of 67% for peptide (C+C*) and 65.6 % for peptide B. The relative molar ratios determined by Edman sequencing were an average of 4.27 (expected ratio of 4) for peptides (C+C*)/B, and 1.49 for peptide C*/C (expected ratio of 1); the seemingly high 49% error in quantification of Lys(Ac) in peptide C* can be attributed to commercial unavailability of its PTH standard. These values compare very favorably with the values obtained by mass spectrometry.

Purification and characterization of three antifungal proteins from cheeseweed (Malva parviflora).

Three potent antimicrobial proteins were purified from cheeseweed (Malva parviflora) seeds. These antimicrobial proteins, named CW-3, CW-4, and CW-5, showed different antimicrobial spectrum and potency compared to the two heterologous antimicrobial proteins (CW-1 and CW-2) purified previously. CW-3 and CW-4 possess antimicrobial activities against Phytophthora infestans (Pi), but not Fusarium graminearum (Fg). A database search indicated that CW-3 shares high homology to cotton vicilin, an abundant seed storage protein. CW-4 shares homology to 2S albumin, another seed storage protein from cotton. CW-5 has antimicrobial activity against Fg, but no activity against Pi was observed at protein concentration up to 50 ppm. Under low salt condition, CW-5 showed potent antimicrobial activity against Fg, but under high salt condition, the antimicrobial activity was drastically diminished. Database search indicated that CW-5 has high homology to a lipid transfer protein from grape. The IC(50) values of the three purified antimicrobial proteins under both low and high salt conditions were determined. The isolation of five antimicrobial proteins for the first time from a single plant source provides further understanding of the plant innate defense system and insight on how plants evolve their complex and complementary antimicrobial system that is important in the early stage of development.

C-TAK1 protein kinase phosphorylates human Cdc25C on serine 216 and promotes 14-3-3 protein binding.

Cdc25C is a dual-specificity protein kinase that controls entry into mitosis by dephosphorylating Cdc2 on both threonine 14 and tyrosine 15. Cdc25C is phosphorylated on serine 216 throughout interphase but not during mitosis. Serine 216 phosphorylation mediates the binding of 14-3-3 protein to Cdc25C, and Cdc25C/14-3-3 complexes are present throughout interphase but not during mitosis. Here we report the cloning of a human kinase denoted C-TAK1 (for Cdc twenty-five C associated protein kinase) that phosphorylates Cdc25C on serine 216 in vitro. C-TAK1 is ubiquitously expressed in human tissues and cell lines and is distinct from the DNA damage checkpoint kinase Chk1, shown previously to phosphorylate Cdc25C on serine 216. Cotransfection of Cdc25C with C-TAK1 resulted in enhanced phosphorylation of Cdc25C on serine 216. In addition, a physical interaction between C-TAK1 and Cdc25C was observed upon transient overexpression in COS-7 cells. Finally, coproduction of Cdc25C and C-TAK1 in bacteria resulted in the stoichiometric phosphorylation of Cdc25C on serine 216 and facilitated 14-3-3 protein binding in vitro. Taken together, these results suggest that one function of C-TAK1 may be to regulate the interactions between Cdc25C and 14-3-3 in vivo by phosphorylating Cdc25C on serine 216.

Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25.

In response to DNA damage, mammalian cells prevent cell cycle progression through the control of critical cell cycle regulators. A human gene was identified that encodes the protein Chk1, a homolog of the Schizosaccharomyces pombe Chk1 protein kinase, which is required for the DNA damage checkpoint. Human Chk1 protein was modified in response to DNA damage. In vitro Chk1 bound to and phosphorylated the dual-specificity protein phosphatases Cdc25A, Cdc25B, and Cdc25C, which control cell cycle transitions by dephosphorylating cyclin-dependent kinases. Chk1 phosphorylates Cdc25C on serine-216. As shown in an accompanying paper by Peng et al. in this issue, serine-216 phosphorylation creates a binding site for 14-3-3 protein and inhibits function of the phosphatase. These results suggest a model whereby in response to DNA damage, Chk1 phosphorylates and inhibits Cdc25C, thus preventing activation of the Cdc2-cyclin B complex and mitotic entry.

Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216.

Human Cdc25C is a dual-specificity protein phosphatase that controls entry into mitosis by dephosphorylating the protein kinase Cdc2. Throughout interphase, but not in mitosis, Cdc25C was phosphorylated on serine-216 and bound to members of the highly conserved and ubiquitously expressed family of 14-3-3 proteins. A mutation preventing phosphorylation of serine-216 abrogated 14-3-3 binding. Conditional overexpression of this mutant perturbed mitotic timing and allowed cells to escape the G2 checkpoint arrest induced by either unreplicated DNA or radiation-induced damage. Chk1, a fission yeast kinase involved in the DNA damage checkpoint response, phosphorylated Cdc25C in vitro on serine-216. These results indicate that serine-216 phosphorylation and 14-3-3 binding negatively regulate Cdc25C and identify Cdc25C as a potential target of checkpoint control in human cells.

Phosphorylation of calmodulin in the first calcium-binding pocket by myosin light chain kinase.

In smooth muscle and specific nonmuscle cells the phosphorylation of the regulatory myosin light chains by myosin light chain kinase (MLCK) is an obligatory step in actin-induced activation of myosin ATPase and subsequent contractile events. We have previously demonstrated that CaM phosphorylated by casein kinase II fails to activate bovine platelet MLCK (Sacks et al. (1992) Biochem. J. 283, 21-24). While myosin light chains are perceived as the only known substrate for MLCK phosphorylation activity, we now show that MLCK phosphorylates CaM. This phosphorylation of CaM is dependent upon the presence of basic peptides such as poly-L-arginine (optimal basic peptide/CaM ratio = 0.08) and is stimulated by saturating [Ca2+] (K0.5 = 16 microM). CaM phosphorylation was inhibited by KT5926, a specific MLCK inhibitor, with a dose-dependency identical to that for inhibition of myosin light chain phosphorylation. Native and MLCK-phosphorylated CaM were indistinguishable in activating MLCK to phosphorylate myosin light chains. Interestingly, MLCK in which the CaM-binding site has been removed is able to phosphorylate CaM in a Ca(2+)-independent manner, suggesting that two CaM molecules bind to intact MLCK simultaneously, one on the inhibitory (pseudosubstrate) domain and one at the catalytic site. CaM phosphorylation by MLCK occurred exclusively on Thr 29 (90%) and Thr 26 (10%) in the first Ca(2+)-binding pocket. In summary, CaM phosphorylation by MLCK differs from CaM phosphorylation catalyzed by other kinases (i.e., the insulin receptor or casein kinase II) in both basic peptide and Ca2+ requirements as well as in the sites of phosphorylation. Further investigations of this model may provide insight into the mechanisms of MLCK activation and substrate recognition.

Identification of insulin-stimulated phosphorylation sites on calmodulin.

Insulin enhances calmodulin phosphorylation in vivo. To determine the insulin-sensitive phosphorylation sites, phosphocalmodulin was immunoprecipitated from Chinese hamster ovary cells expressing human insulin receptors (CHO/IR). Calmodulin was constitutively phosphorylated on serine, threonine, and tyrosine residues, and insulin enhanced phosphate incorporation on serine and tyrosine residues. Phosphocalmodulin immunoprecipitated from control and insulin-treated CHO/IR cells, and calmodulin phosphorylated in vitro by the insulin receptor kinase and casein kinase II were resolved by two-dimensional phosphopeptide mapping. Several common phosphopeptides were detected. The phosphopeptides from the in vitro maps were eluted and phosphoamino acid analysis, manual sequencing, strong cation exchange chromatography, and additional proteolysis were performed. This strategy demonstrated that Tyr-99 and Tyr-138 were phosphorylated in vitro by the insulin receptor kinase and Thr-79, Ser-81, Ser-101 and Thr-117 were phosphorylated by casein kinase II. In vivo phosphorylation sites were identified by comigration of phosphopeptides on two-dimensional maps with phosphopeptides derived from calmodulin phosphorylated in vitro and by phosphoamino acid analysis. This approach revealed that Tyr-99 and Tyr-138 of calmodulin were phosphorylated in CHO/IR cells in response to insulin. Additional sites remain to be identified. The identification of the insulin-stimulated in vivo tyrosine phosphorylation sites should facilitate the elucidation of the physiological role of phosphocal-modulin.

Facile, in situ matrix-assisted laser desorption ionization-mass spectrometry analysis and assignment of disulfide pairings in heteropeptide molecules.

During a routine analysis of disulfide-linked synthetic heterodipeptides by matrix-assisted laser desorption ionization (MALDI) mass spectrometry with linear detection we observed not only the expected mass of the dipeptide, but also the mass of the individual constituent monomer peptides. This was surprising because the peptide was purified as an intact dipeptide and no overt attempt was made to reduce the disulfide linkage before mass analysis. In contrast, analysis of the same sample by electrospray ionization mass spectrometry gave the mass of the dipeptide only. To investigate this further, two additional model heterodipeptides were prepared and all three were used to systematically study several matrix-assisted laser desorption ionization mass spectrometry parameters. These parameters were three different matrices (alpha-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, and sinapinic acid), both positive and negative modes of detection, and varying the acceleration voltage from 5 to 20 kV. Except for the sinapinic acid matrix where poor-quality spectra were obtained, all three model heterodipeptides fragmented under the tested conditions in a manner consistent with the cleavage of disulfide bonds, although the absolute level was sample dependent. The precise mechanism of disulfide cleavage during analysis is unknown, but the cleavage we observed appears to originate during the initial ionization event. Because the MALDI process involves irradiating samples with a laser, the fragmentation of disulfide-linked peptides that we observe bears some resemblance to light-induced homolytic cleavage of aqueous solutions of the amino acid cystine, although other mechanisms for fragmentation are also possible.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine-phosphorylated calmodulin has reduced biological activity.

Calmodulin is phosphorylated by the purified insulin receptor on tyrosine residues with a maximum stoichiometry of 1 mol phosphate/mol of calmodulin. Isolated tryptic phosphopeptides were sequenced by manual Edman degradation and demonstrated that calmodulin is equally phosphorylated on tyrosine 99 and tyrosine 138. Phosphorylated calmodulin has a decreased affinity (K0.5 = 4.2 nM) for the 63-kDa isozyme of cyclic nucleotide phosphodiesterase compared to nonphosphorylated calmodulin (K0.5 = 2.1 nM). The K0.5 for Ca2+ is marginally increased from 2.8 to 3.2 microM in the presence of phosphotyrosyl calmodulin. The effect of the calmodulin antagonist, mastoparan, was investigated to determine whether mastoparan would differentially inhibit calmodulin- or phosphocalmodulin-dependent enzyme activity. The IC50 of mastoparan is fourfold lower for phosphotyrosyl calmodulin compared to nonphosphorylated calmodulin. Phosphorylation of calmodulin may provide a mechanism for the differential regulation of calmodulin-dependent enzymes. These observations further support a potentially important regulatory function of calmodulin phosphorylation in signal transduction.

Disulfide bonds required to assemble functional von Willebrand factor multimers.

The hemostatic functions of human von Willebrand Factor (vWF) depend on the normal assembly of disulfide-linked multimers from approximately 250-kDa subunits. Subunits initially form dimers through disulfide bonds near the COOH terminus. Dimers then form multimers through disulfide bonds near the NH2 terminus of each subunit. Previous studies of plasma vWF and recombinant vWF fragments indicate that 1 or more of the Cys residues at position 459, 462, and 464 form intersubunit disulfide bonds. No evidence has been reported that vWF multimer formation involves additional intersubunit bonds. To probe the disulfide bond requirements for multimer formation, mutant vWF proteins were expressed in which all 3 Cys residues at positions 459, 462, and 464 were changed to either Gly or Ala. Surprisingly, none of these cysteines appears to be necessary for efficient multimer assembly. Furthermore, recombinant vWF with Gly or Ala at all three positions induces platelet aggregation in the presence of ristocetin and binds to platelet glycoprotein Ib, factor VIII, and collagen in a manner similar to wild-type recombinant vWF. These results suggest that other intersubunit disulfide bonds must exist. Direct evidence for such a bond was obtained by characterization of tryptic fragments of vWF. By Edman degradation, amino acid composition, and mass spectrometry, a disulfide bond was demonstrated between Cys379 residues of adjacent vWF subunits. Thus, intersubunit disulfide bonds involving Cys379 and 1 or more of the Cys residues at positions 459, 462, and 464 connect the NH2-terminal ends of the vWF subunits in a parallel orientation.

Phosphorylation of the insulin receptor substrate IRS-1 by casein kinase II.

IRS-1, a principal substrate of the insulin receptor, is phosphorylated on serine, threonine, and tyrosine residues in a variety of tissues during insulin stimulation. Casein kinase II, an insulin-sensitive serine/threonine kinase, catalyzed the in vitro incorporation of 1 to 2 mol of phosphate/mol of recombinant rat IRS-1. Two-dimensional phosphopeptide mapping of IRS-1 phosphorylated by casein kinase II in vitro and IRS-1 immunoprecipitated from intact Chinese hamster ovary cells demonstrated multiple common phosphopeptides, suggesting that overexpressed IRS-1 is a substrate for casein kinase II in these cells. Moreover, the common phosphopeptides that appeared to be insulin-sensitive in intact cells comprised 22% of casein kinase II-catalyzed 32P incorporation into IRS-1 in vitro. These data suggest that casein kinase II mediates a portion of the insulin-stimulated serine/threonine phosphorylation of overexpressed IRS-1 in vivo. By using phosphoamino acid analysis, strong cation exchange analysis, manual Edman degradation, and automated amino acid sequencing, Thr-502 was identified as the major casein kinase II-catalyzed phosphorylation site in rat IRS-1. Furthermore, Ser-99, an additional site labeled at low yield, appeared to be contained in an insulin-sensitive phosphopeptide. Thus, casein kinase II-catalyzed phosphorylation of IRS-1 may be a component of the intracellular insulin signalling cascade.

Semi-automated chromatographic procedure for the isolation of acetylated N-terminal fragments from protein digests.

Several published procedures have been combined to develop a general strategy for the specific identification and isolation of the acetylated-N-terminal fragment from all other proteolytic fragments. This ruse can be divided into four steps: (i) succinylation of the substrate to block lysine NH2 groups; (ii) enzymatic digestion of the modified protein; (iii) automated phenylisothiocyanate derivatization of the protease derived fragments to block newly generated "free" N-termini; and (iv) reversed-phase high-performance liquid chromatography with on-line photodiode array spectroscopy. The individual phenylthiocarbamyl-peptide species exhibit an increased reversed-phase retention time and a greater UV (210-297 nm) profile compared to the corresponding control (-phenylisothiocyanate) digest. The N-terminal acetylated fragment shows neither a retention time shift nor an augmented UV profile. To validate each process step, synthetic peptides and acetylated-N-terminal proteins of known sequence were used as test samples. The desired fragment was isolated from three proteins and positively identified by electrospray mass spectrometry and amino acid composition. Proteins with other N-terminal blocking groups should be amenable to this procedure.

Chromatographic analysis of tropomyosins from rabbit skeletal, chicken gizzard and earthworm muscle.

Tropomyosins from rabbit skeletal, chicken gizzard and earthworm muscle all exist as dimeric, ca. 100% alpha-helical coiled-coil species in benign media. Two major tropomyosin isoforms from each muscle source have been identified and can be conveniently designated alpha (fast) and beta (slow) based on electrophoretic mobility under denaturing conditions. The ratio of alpha to beta chains is ca. 3-4:1 for rabbit skeletal and ca. 1:1 for chicken gizzard and earthworm tropomyosins. Each chain from the former two muscle sources has been sequenced, thus providing a molecular basis for interpreting the in vivo population of homo- and hetero-dimers. The characteristics of each purified tropomyosin in weak-anion exchange, strong-cation exchange and reversed-phase high-performance liquid chromatography are described. Binding to and/or elution from the reversed-phase matrix results in dissociation into highly helical monomeric chains. This mode of chromatography separates the alpha and beta chains of earthworm and chicken gizzard tropomyosins, but not those of the rabbit protein. Both anion- and cation-exchange chromatography use mild (benign) elution conditions under which the native, in vivo dimer population should be preserved. Only the rabbit protein exhibited peak separation on the anion-exchange resin, with peak assignment corresponding to the known molecular organization of homo- and hetero-dimers. In strong cation-exchange analysis, all three tropomyosins exhibit a chromatographic transition near pH 6.5, possibly the result of histidine(s) titration. Collectively, the chromatographic data confirm the present understanding of the in vivo mixture of dimers for tropomyosin from rabbit skeletal and chicken gizzard. It is concluded that native earthworm tropomyosin exists predominantly as an alpha beta hetero-dimer.

Automated phenylthiocarbamyl amino acid analysis of carboxypeptidase/aminopeptidase digests and acid hydrolysates.

A fully automated exopeptidase digestion procedure for the partial determination of N- and C-terminal peptide/protein sequence is described. The digestion of various substrates with aminopeptidase M, carboxypeptidase A, P or Y was accomplished with the Varian 9090 autosampler's robotic automix routines. The released free amino acids, in addition to free amino acids from acid hydrolysates, were derivatized with phenylisothiocyanate in an automated fashion and subsequently chromatographed on a C18 column for separation and quantitation. The advantages of automating this precolumn phenylisothiocyanate derivatization are the virtual elimination of sample manipulation errors and very reproducible data due to the precise control of the reaction conditions both of which, facilitate the interpretation of the exopeptidase reaction kinetic data.

In situ chemical cleavage of proteins immobilized to glass-fiber and polyvinylidenedifluoride membranes: cleavage at tryptophan residues with 2-(2'-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine to obtain internal amino acid sequence.

Proteins immobilized to glass-fiber supports and polyvinylidenedifluoride membranes are cleaved in situ with a tryptophan residue-specific reagent, 2-(2'-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine. Protein fragments can be eluted from polyvinylidenedifluoride membranes after in situ digestion and electrophoresed and electroblotted to new polyvinylidenedifluoride membranes for subsequent sequence determination of selected bands. Five proteins (bovine serum albumin, ovalbumin, beta-casein, beta-lactoglobulin, and myoglobin) of known sequence containing one to three tryptophan residues each were used as model substrates to evaluate the specificity and extent of cleavage. Under the conditions used, the reaction is rapid and exhibits a high degree of specificity for tryptophan residues since N-terminal sequence analysis of the digested, immobilized samples yields the expected, newly generated N-termini. No detectable cleavage occurred at nontryptophan residues nor at acid labile aspartic acid-proline peptide bonds. Solution cleavage reactions were also performed and the resulting digest was analyzed by gel electrophoresis. We found a positive correlation between the solution and in situ cleavage reactions, in that, proteins which show cleavage in the solution reaction also yield internal sequence data after in situ digestion. Since tryptophan occurs with low frequency in proteins, this cleavage reaction has the potential to generate a small number of fragments and in favorable circumstances allow sequence analysis of the unfractionated reaction mixture. In addition, the sequence obtainable after, but not before, in situ digestion can be used as an indication that the N-terminus of the protein is blocked.

Strong-cation-exchange sulfoethyl aspartamide chromatography for peptide mapping of Staphylococcus aureus V8 protein digests.

In two recent reports (D. L. Crimmins, J. Gorka, R. S. Thoma, and B. D. Schwartz (1988) J. Chromatogr. 443, 63-71; A. J. Alpert and P. C. Andrews (1988) J. Chromatogr. 443, 85-96) a sulfoethyl aspartamide column was shown to efficiently analyze peptides less than 25 residues in length which differ in the number of nominal positive charges at pH 3.0. In particular, the elution order for a series of distinct peptides ranging in nominal charge from +1 to +7 was found to be monotonic in nature indicating that separation was primarily via a cation-exchange mechanism. The present study employs this chromatographic system to isolate and characterize major fragments of proteolytic digests. Six commercially available proteins of known sequence (myoglobin, beta-casein, concanavalin A, carbonic anhydrase, lentil lectin, and enolase) were digested with Staphylococcus aureus V8 to generate peptide fragments. The resulting mixture was chromatographed on a sulfoethyl aspartamide column to isolate major fragments which were then subjected to amino acid analysis and N-terminal sequencing. With complete proteolysis (i.e., peptide fragments terminating in either an aspartic or a glutamic acid) separation of the fragments should result from the sum of histidine, lysine, and arginine residues contained in each fragment. Most of the peptide fragments eluted at the expected time on the sulfoethyl aspartamide column. Those fragments with anomalous behavior resulted from incomplete cleavage or cleavage at nonacidic residues or were greater than 35 residues in length. Each proteolytic digest was also analyzed by standard reverse-phase C4 chromatography to compare the peptide maps for these two distinct chromatographic modes.

Peptide characterization with a sulfoethyl aspartamide column.

A strong cation-exchange (SCX) high-performance liquid chromatography column (sulfoethyl aspartamide, 200 x 4.6 mm) was used to analyze more than 50 peptides, ranging in length from 5 to 20 residues. These data show that the elution positions of the peptides increase monotonically with the number of positively charged residues. [A 60-min linear gradient of 0 to 100% eluent B at 1 ml/min was used, where eluent A is 5 mM phosphate (pH 3.0)-acetonitrile (75:25) and eluent B is eluent A + 0.5 M sodium chloride.] A comparison of SCX with a standard C18 reversed-phase (RP) column [60-min linear gradient of 0 to 60% B at 1 ml/min, where eluent A is 0.1% trifluoroacetic acid (TFA), and eluent B is 0.095% TFA-acetonitrile (10:90)] further demonstrates the utility of SCX in peptide characterization. SCX separated an (Arg)3-containing peptide from the Arg-deleted peptide while RP could not. In addition, SCX and RP resolved the methionine oxidation products of ACTH (4-10) (RP: Met [O] less than Met [O2] less than Met; SCX; Met [O] less than Met less than Met [O2]), suggesting a mixed-mode mechanism for the ion-exchange system. Finally, SCX separated the sulfated and non-sulfated forms of cholecystokinin (26-33) and Leu-enkephalin as well as the N-terminal acetylated forms of neurotensin (8-13) and angiotensinogen (1-14) from the respective unmodified peptides.