Capillary HPLC Research Articles

Fast preparation of polystyrene-based monolith using microwave irradiation for micro-column separation

The conventional analytical HPLC was successfully developed for micro-column separation by using a simple eluate splitting system, a self-preparation of monolithic column and an on-capillary column detector in our laboratory. A typical polystyrene-based monolith was quickly prepared inside the fused-silica capillary, which in situ polymerization was carried out in 10 min by microwave irradiation. The reactant solution consisted of styrene (ST) as a functional monomer, divinylbenzene (DVB) as a cross-linking agent, toluene and isooctane as porogenic solvents, and azobisisobutyronitrile (AIBN) as an initiator. The monolith was proved to form in the center of the capillary and adhered to the column inner wall by the scanning electron micrograph. Its chromatographic behaviors were evaluated in detail by varying the flow rate and percentage of mobile phases, and under the optimal condition, baseline separation of the model analytes including thiourea, benzene, toluene, ethylbenzene was obtained with a highest theoretical plate number near 11,290 N/m by the developed capillary HPLC. Furthermore, the stability and porosity of the prepared monolith were systematically investigated by a simple flow method.

Determination of aromatic hydrocarbons in petroleum jet fuels by capillary gas chromatography and high-performance liquid chromatography

About a hundred hydrocarbon compounds have been identified as components of petroleum jet fuel by capillary gas chromatography and high-performance liquid chromatography. In gas chromatography stuides, glass capillary columns over 100 m long with polysiloxane OV-101 have been used as the stationary phase. In liquid chromatography, 150-mm-long columns with a separation ability of about 60 000 theoretical plates meter of length have been used to separate partitioned fractions of aromatic hydrocarbons from petroleum jet fuels of various origins.

Enantiomeric Resolution of Benzoxazolinonic Aminoalcohols, Potential Adrenergic Ligands, by LC and CE

Benzoxazolinone amino alcohols are chiral derivatives that carry one asymmetric center. Both capillary electrophoresis and high-performance liquid chromatography (LC) techniques were used to separate enantiomers with high resolution and thus permitted the determination of enantiomeric excess. Highly sulfated and anionic cyclodextrins were used as chiral selectors under CE conditions. Amylose and cellulose chiral stationary phases were used for LC for the enantioseparation of five racemic compounds. Separations were carried out on a Chiralpak AS column with various polar organic modifiers. The HPLC method appeared more to be more sensitive and was subsequently validated in terms of limits of detection and quantification. LC detection was based on UV and evaporating light scattering.

Proteome analysis of silkworm 1. Fat body

Proteome analysis of silkworm (Bombyx mori L.) fat bodies was performed on the fifth-instar day-3 larva stage and the expression of proteins separated on two-dimensional polyacrylamide gels was tracked from the fourth-instar day-1 larva stage to the stage before adult moth. Proteins on the 2D-gels were excised manually and treated with 4-vinylpyridine for alkylation. Each spot was analyzed by capillary high-performance liquid chromatography coupled with ion-trap mass spectrometry after proteolysis using trypsin. In the previous report1), the amino acid sequence database obtained from the Drosophila genome was used for the protein identification. This analysis used the amino acid sequence data elucidated from the silkworm genome. Additionally, protein expression in fat bodies was compared with that at other stages and in other tissues described in the silkworm proteome database (http://kaiko2ddb.dna.affrc.go.jp/cgi-bin/search_2DDB.cgi). Several unidentified proteins from the previous report1) were identified with high Mascot scores. Induction and reduction of proteins during metamorphosis were observed as changes in spot concentration on sequential 2D-gels on the web.

Proteomic analysis of tumor necrosis factor-α resistant human breast cancer cells reveals a MEK5/Erk5-mediated epithelial-mesenchymal transition phenotype

IntroductionDespite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-α. Additionally, the relationship between TNF-α resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated.MethodsTo compare differences in the proteome of the TNF-α resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-α resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT.ResultsProteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (δ-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression.ConclusionsThis study demonstrates that MEK5 over-expression promotes a TNF-α resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.

DETERMINATION OF POLYNUCLEAR AROMATIC HYDROCARBONS (PAHs) IN SEWAGE SLUDGE BY MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY AND HPLC‐FLUORESCENCE DETECTION: A COMPARATIVE STUDY

Two different methods, based on micellar electrokinetic capillary chromatography (MECC‐DA) and high performance liquid chromatography (HPLC)‐fluorescence detection, were studied to separate and determine 12 polynuclear aromatic hydrocarbons (PAHs) of 16 PAHs recommended by the Environmental Protection Agency (EPA) in sewage sludge. β‐cyclodextrin was used as a micelle precursor in a sodium borate buffer solution which included the surfactant sodium dodecyl sulphate (SDS) and the acetonitrile dissolvent. The following parameters: temperature, voltage, pH, concentrations of buffer solution and injection time, were optimized by an experimental design (Nemrodw programme). Furthermore, HPLC‐fluorescence detection and separation conditions are reported. The mobile phase was acetonitrile‐water in different percentages. Precision, accuracy, linearity and detection and quantification limits were determined. This method was applied in the analysis of sewage sludge from a wastewater depuration station in Crispijana (Álava, Spain). In this case, it is necessary to carry out a solid phase extraction step for its determination. The method was validated by the analysis of a certificated sample of LGC 6182, and finally samples of sewage sludge were analysed weekly for two years, obtaining in all cases results under the legal limits for the use of sewage sludge in agriculture.

In vitro nonenzymatic glycation of guanosine 5′-triphosphate by dihydroxyacetone phosphate

Dihydroxyacetone phosphate (DHAP) is a glycolytic intermediate that has been found to be significantly elevated in the erythrocytes of diabetic patients and patients with triosephosphate isomerase deficiency. DHAP spontaneously breaks down to methylglyoxal, a potent glycating agent that reacts with proteins and nucleic acids in vivo to form advanced glycation endproducts (AGEs). Like methylglyoxal, DHAP itself is also a glycating metabolite, capable of condensing with proteins and altering their structure or function. The objective of this investigation was to evaluate the susceptibility of nucleotides to nonenzymatic attack by DHAP, and to determine the factors influencing the rate and extent of nucleotide glycation by this sugar. Of the four nucleotide triphosphates (ATP, CTP, GTP and UTP) that were studied, only GTP was reactive, forming a wide range of UV and fluorescent products with DHAP. Increases in temperature and nucleotide concentration enhanced the rate and extent of GTP glycation by DHAP and promoted the heterogeneity of AGEs. Capillary electrophoresis, HPLC, and mass spectrometry allowed for a thorough analysis of the glycated products and demonstrated that the reaction of DHAP with GTP occurred via the classical Amadori pathway.

Comprehensive proteomics of Methylobacterium extorquens AM1 metabolism under single carbon and nonmethylotrophic conditions

In order to validate a gel free quantitative proteomics assay for the model methylotrophic bacterium Methylobacterium extorquens AM1, we examined the M. extorquens AM1 proteome under single carbon (methanol) and multicarbon (succinate) growth, conditions that have been studied for decades and for which extensive corroborative data have been compiled. In total, 4447 proteins from a database containing 7556 putative ORFs from M. extorquens AM1 could be identified with two or more peptide sequences, corresponding to a qualitative proteome coverage of 58%. Statistically significant nonzero (log(2) scale) differential abundance ratios of methanol/succinate could be detected for 317 proteins using summed ion intensity measurements and 585 proteins using spectral counting, at a q-value cut-off of 0.01, a measure of false discovery rate. The results were compared to recent microarray studies performed under equivalent chemostat conditions. The M. extorquens AM1 studies demonstrated the feasibility of scaling up the multidimensional capillary HPLC MS/MS approach to a prokaryotic organism with a proteome more than three times the size of microbes we have investigated previously, while maintaining a high degree of proteome coverage and reliable quantitative abundance ratios.

Determination of phenolic acids by capillary zone electrophoresis and HPLC

Abstract Selected phenolic acids are determined by capillary zone electrophoresis and HPLC, each using UV detection. The optimised CZE background electrolyte contained 50 mM acetic acid, 95 mM 6-aminocaproic acid, 0.1% polyacrylamide, 1% polyvinylpyrrolidone, and 10% methanol. Twelve phenolic acids (gallic, p-hydroxybenzoic, 3,4-dihydroxybenzoic, vanillic, syringic, o-coumaric, p-coumaric, caffeic, sinapic, ferulic, salicylic and chlorogenic) were separated within 10 minutes. Chromatographic separation of these phenolic acids was carried out on an Eclipse XBD C8 column using a mobile phase gradient (acetonitrile / methanol / water / 0.1% phosphoric acid); all were separated within 25 minutes. Electrophoretic and chromatographic determinations of ferulic and chlorogenic acids were compared on barley, malt, and potato samples. The methods’ characteristics were: linearity (1–20 mg ml and 0.2–4 mg ml−1), accuracy (recovery 94 ± 5% and 96 ± 4%), intra-assay repeatability (4.1% and 3.5%), and detection limit (0.2 and 0.02 mg ml−1).

Comparison of Two-Dimensional Fractionation Techniques for Shotgun Proteomics

Two-dimensional (2D) fractionation is a commonly used tool to increase dynamic range and proteome coverage for bottom-up, shotgun proteomics. However, there are few reports comparing the relative separation efficiencies of 2D methodologies using low-microgram sample quantities. In order to systematically evaluate 2D separation techniques, we fractionated microgram quantities of E. coli protein extract by seven different methods. The first dimension of separation was performed with either reversed-phase high-pressure liquid chromatography (RP-HPLC), gel electrophoresis (SDS-PAGE), or strong cation exchange (SCX-HPLC). The second dimension consisted of a standard reversed-phase capillary HPLC coupled to an electrospray ionization quadrupole time-of-flight mass spectrometer for tandem mass spectrometric analysis. The overall performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The protein-level RP-HPLC and the high-pH RP-HPLC peptide-level separations performed the best, identifying 281 and 266 proteins, respectively. The online pH variance SCX and the SDS-PAGE returned modest performances with 178 and 139 proteins identified, respectively. The offline SCX had the worst performance with 81 proteins identified. We also examined various chromatographic factors that contribute to separation efficiency, including resolving power, orthogonality, and sample loss.

Dietary n-3 Polyunsaturated Fatty Acids Enhance Hormone Ablation Therapy in Androgen-Dependent Prostate Cancer

Hormone ablation therapy typically causes regression of prostate cancer and represents an important means of treating this disease, particularly after metastasis. However, hormone therapy inevitably loses its effectiveness as tumors become androgen-independent, and this conversion often leads to death because of subsequent poor responses to other forms of treatment. Because environmental factors, such as diet, have been strongly linked to prostate cancer, we examined the affects of dietary polyunsaturated fatty acids (PUFAs; at 1.5 wt%) on growth of androgen-dependent (CWR22) and androgen-independent (CWR22R) human prostatic cancer xenografts, the acute response of CWR22 tumors to ablation therapy, and their progression to androgen independence. Significant diet-induced changes in tumor n-3 or n-6 PUFA content had no affect on CWR22 or CWR22R tumors growing with or without androgen support, respectively. However, dietary changes that increased tumor eicosapentaenoic acid and linoleic acid content enhanced responses to ablation therapy, measured by cancer cell apoptosis and mitosis. In addition, relapse to androgen-independent growth (measured by renewed increases in tumor volume and serum prostate-specific antigen after ablation) positively correlated with tumor arachidonic acid content. There was no correlation between expression of 15-lipoxygenase isozymes or their products and tumor growth or responses to ablation. In conclusion, dietary n-3 PUFA may enhance the response of prostate cancer to ablation therapy and retard progression to androgen-independent growth by altering tumor PUFA content.

Regulation of Intra-S Phase Checkpoint by Ionizing Radiation (IR)-dependent and IR-independent Phosphorylation of SMC3

Structure maintenance of chromosome 1 (SMC1) is phosphorylated by ataxia telangiectasia-mutated (ATM) in response to ionizing radiation (IR) to activate intra-S phase checkpoint. A role of CK2 in DNA damage response has been implicated in many previous works, but the molecular mechanism for its activation is not clear. In the present work, we report that SMC3 is phosphorylated at Ser-1067 and Ser-1083 in vivo. Ser-1083 phosphorylation is IR-inducible, depends on ATM and Nijmegen breakage syndrome 1 (NBS1), and is required for intra-S phase checkpoint. Interestingly, Ser-1067 phosphorylation is constitutive and is not induced by IR but also affects intra-S phase checkpoint. Phosphorylation of Ser-1083 is weakened in cells expressing S1067A mutant, suggesting interplay between Ser-1067 and Ser-1083 phosphorylation in DNA damage response. Consistently, small interfering RNA knockdown of CK2 leads to attenuated phosphorylation of Ser-1067 as well as intra-S phase checkpoint defect. Our data provide evidence that phosphorylation of a core cohesin subunit SMC3 by ATM plays an important role in DNA damage response and suggest that a constitutive phosphorylation by CK2 may affect intra-S phase checkpoint by modulating SMC3 phosphorylation by ATM.

Ultra-Fast Separation of Biomolecules Using Superficially Porous Silica Particles – Poroshell

Ultra fast separation of large biomolecules is always a challenge for chemists because of slow mass transfer rate of large biomolecules. Superficially porous reversed-phase HPLC packings have taken their place in the scientific toolkit as the optimum particle for ultra-fast separations of proteins and polypeptides [1]. Versatility in this arena has been increased due to the development of sterically protected C18, C8 and C3 bonded phases for low pH application and bidentate C18 for high pH application. The new column formats such as capillary HPLC column not only increase the speed of the separation, but also dramatically increase the sensitivity of the separation.

A Revised Role for P-Glycoprotein in the Brain Distribution of Dexamethasone, Cortisol, and Corticosterone in Wild-Type and ABCB1A/B-Deficient Mice

The ABCB1-type multidrug resistance efflux transporter P-glycoprotein (P-gp) has been hypothesized to regulate hypothalamic-pituitary-adrenal axis activity by limiting the access of glucocorticoids to the brain. In vivo systemic administration studies using P-gp-deficient mice have shown increased glucocorticoid entry to the brain compared with wild-type controls. However, these studies did not control for the presence of radiolabeled drug in the capillaries, verify an intact blood-brain barrier, or confirm stability of the glucocorticoids used. In the present study, an in situ brain perfusion method, coupled with capillary depletion and HPLC analyses, was used to quantify brain uptake of [3H]dexa-methasone, [3H]cortisol, and [3H]corticosterone in P-gp-deficient and control mice. A vascular marker was included in these experiments. The results show that brain uptake of [3H]dexamethasone was increased in the frontal cortex, hippocampus, hypothalamus, and cerebellum of P-gp-deficient mice compared with wild-type controls. Brain uptake of [3H]cortisol was increased in the hypothalamus of P-gp-deficient mice compared with wild-type controls, but no differences were detected in other regions. Brain uptake of [3H]corticosterone was not increased in P-gp-deficient mice compared with wild-type controls in any brain areas. After our systemic administration of the same radiolabeled glucocorticoids, HPLC analysis of plasma samples identified additional radiolabeled components, likely to be metabolites. This could explain previous findings from systemic administration studies, showing an effect of P-gp not only for dexamethasone and cortisol, but also for corticosterone. This in situ study highlights the different affinities of dexamethasone, cortisol, and corticosterone for P-gp, and suggests that the entry of the endogenous glucocorticoids into the mouse brain is not tightly regulated by P-gp. Therefore, our current understanding of the role of P-gp in hypothalamic-pituitary-adrenal regulation in mice requires revision.

Increased Throughput and Reduced Carryover of Mass Spectrometry-Based Proteomics Using a High-Efficiency Nonsplit Nanoflow Parallel Dual-Column Capillary HPLC System

We report a new design of a fully automated, high-efficiency parallel nonsplit nanoflow capillary HPLC system, coupled on-line with linear ion trap (LTQ) and high performance nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoESI LTQ-FTICR MS). The system, intended for high-throughput proteome analysis of complex protein mixtures, notably serum and plasma, consists of two reversed-phase trap columns for large volume sample injection with high speed sample loading and desalting and two reversed-phase analytical capillary columns. Through a nanoscale two-position, 10-port switching valve, the whole system is terminated by a 10 microm i.d. of nanoemitter mounted on the nanoelectrospray source in front of the sampling cone of the LTQ-FTICR MS. Gradient elution to both nanoflow-rate capillary columns is simultaneously delivered by a single HPLC system via two independent binary gradient pump systems. The parallel capillary column approach eliminates the time delays for column regeneration/equilibration since one capillary column is used for separating the sample mixtures and delivering the separated fractions to the MS, while the other capillary column is being regenerated and equilibrated. The reproducibility of retention time and peak intensity of the present automated parallel nanoflow-rate capillary HPLC system is comparable to that obtained using a single column configuration. Replicate injections of tryptic digests indicated that this system provided good reproducibility of retention time and peak area on both columns with average CV values of less than 1.08% and 7.04%, respectively. Throughput was increased to 100% for 2-h LC-MS analysis compared to the single capillary column LC-MS pipeline. Application of this system is demonstrated in a plasma proteomic study. A total of 312 868 MSMS events were acquired and 1564 proteins identified with high confidence (Protein Prophet > or = 0.9, and peptides matched > or = 2). Comparison of a series of plasma fractions run using the single-column LC-MS versus the parallel-column LC-MS demonstrated that parallel-column LC-MS system significantly reduced the sample carryover, improved MS data quality and increased the number of MS/MS sequence scan events.

Gold microspheres modified with octadecanethiol for capillary liquid chromatography

Monodispersed spherical gold particles synthesized and modified with n-octadecanethiol (C18-Au) were packed into a 100 μm I.D. capillary column and tested in capillary high-performance liquid chromatography (μHPLC). To the best of our knowledge, this represents the first report on the actual use of micron gold particles as stationary phase in a packed column μHPLC. As measured by scanning electron microscopy, the average diameter of these gold microspheres was 3.5 μm and the surface area, average pore diameter, and average pore volume were 49.4 m 2/g, 4.8 nm, and 0.12 cm 3/g, respectively. The retention behavior of neutral compounds on the n-octadecanethiol-modified gold microspheres was investigated by separating a mixture of small organic compounds using μHPLC. The results from the experiments show that C18-Au behaves basically as a reversed phase. The test of chemical stability of the C18-Au stationary phase under alkaline condition demonstrates that it is stable with the flushing of a mobile phase at pH 12 for at least 140 h. The C18-Au particles are also mechanically strong enough to withstand pressure up to 52 MPa. The preliminary results in this work prove the feasibility of the fabrication of C18-Au micron particles as a novel stationary phase for packed column μHPLC.

Capillary liquid chromatography–mass spectrometry for the rapid identification and quantification of almond flavonoids

A rapid negative ion ESI high-performance capillary liquid chromatography–mass spectrometry method was developed to identify and quantify flavonoids (e.g., flavanols, flavonols, flavanones and glycosides). Fifteen standards and two varieties of almond skin extract powder (Carmel and Nonpareil) were used to demonstrate the chromatographic separation, reproducibility and accuracy of the method that employed a 150 mm × 0.3 mm ChromXP 3C18-EP-120 column. All standards eluted in less than 10 min, providing a 9–12× reduction in analysis time compared to existing methods (90–120 min). However, isomers (e.g., catechin/epicatechin and galactosides/glucosides) were not resolved and, therefore, identified and quantified collectively. RSDs for retention time and peak area reproducibility (mass spectrometry data) were <0.5% and <5.0%, respectively. Peak area reproducibility was greatly improved (from a RSD > 10%) after the implementation of a low-flow metal needle in the ESI source. Quantitation by mass spectrometry also afforded a % error less than 5% for most compounds.

Automated Neutral Loss and Data Dependent Energy Resolved “Pseudo MS3” for the Targeted Identification, Characterization and Quantitative Analysis of Methionine-Containing Peptides

A strategy involving the fixed-charge sulfonium ion derivatization, stable isotope labeling, capillary high- performance liquid chromatography and automated data dependent neutral loss scan mode tandem mass spectrometry (MS/MS) and "pseudo multiple mass spectrometry (MS3)" product ion scans in a triple quadrupole mass spectrometer has been developed for the "targeted" gas-phase identification, characterization and quantitative analysis of low abundance methionine-containing peptides present within complex protein digests. Selective gas-phase "enrichment" and identification is performed via neutral loss scan mode MS/MS, by low energy collision-induced dissociation of the derivatized methionine side chain, resulting in the formation of a single characteristic product ion. Structural characterization of identified peptides is then achieved by automatically subjecting the characteristic neutral loss product ion to further dissociation by data dependent product ion scan mode pseudo MS3 under higher collision energy conditions. Quantitative analysis is achieved by measurement of the abundances of characteristic product ions formed by sequential neutral loss scan mode MS/MS experiments from "light" (12C) and "heavy" (13C) stable isotope encoded fixed-charge derivatized peptides. In contrast to MS-based quantitative analysis strategies, the neutral loss scan mode MS/MS method employed here was able to achieve accurate quantification for individual peptides at levels as low as 100 fmol and at abundance ratios ranging from 0.1 to 10, present within a complex protein digest.

Cdc28 and Cdc14 Control Stability of the Anaphase-promoting Complex Inhibitor Acm1

The anaphase-promoting complex (APC) regulates the eukaryotic cell cycle by targeting specific proteins for proteasomal degradation. Its activity must be strictly controlled to ensure proper cell cycle progression. The co-activator proteins Cdc20 and Cdh1 are required for APC activity and are important regulatory targets. Recently, budding yeast Acm1 was identified as a Cdh1 binding partner and APC(Cdh1) inhibitor. Acm1 disappears in late mitosis when APC(Cdh1) becomes active and contains conserved degron-like sequences common to APC substrates, suggesting it could be both an inhibitor and substrate. Surprisingly, we found that Acm1 proteolysis is independent of APC. A major determinant of Acm1 stability is phosphorylation at consensus cyclin-dependent kinase sites. Acm1 is a substrate of Cdc28 cyclin-dependent kinase and Cdc14 phosphatase both in vivo and in vitro. Mutation of Cdc28 phosphorylation sites or conditional inactivation of Cdc28 destabilizes Acm1. In contrast, inactivation of Cdc14 prevents Acm1 dephosphorylation and proteolysis. Cdc28 stabilizes Acm1 in part by promoting binding of the 14-3-3 proteins Bmh1 and Bmh2. We conclude that the opposing actions of Cdc28 and Cdc14 are primary factors limiting Acm1 to the interval from G(1)/S to late mitosis and are capable of establishing APC-independent expression patterns similar to APC substrates.

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.