Radiolytic modification of sulfur-containing amino acid residues in model peptides: fundamental studies for protein footprinting

Protein footprinting based on hydroxyl radical-mediated modification and quantitative mass spectroscopic analysis is a proven technique for examining protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural analysis depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-containing amino acids are oxidized readily and the mechanisms of oxidation are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here we have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides containing cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidation using gamma-rays or synchrotron X-rays and have compared these results to those expected from oxidation mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are observed. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidation is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the gamma-carbon. Due to the high reactivity of sulfur-containing amino acids, the extent of oxidation is easily influenced by secondary oxidation events or the presence of redox reagents used in standard proteolytic digestions; when these are accounted for, a reactivity order of cysteine > methionine approximately tryptophan > cystine is observed.     

Millisecond radiolytic modification of peptides by synchrotron X-rays identified by mass spectrometry.

Radiolysis of peptide and protein solutions with high-energy X-ray beams induces stable, covalent modifications of amino acid residues that are useful for synchrotron protein footprinting. A series of 5-14 amino acid residue peptides of varied sequences were selected to study their synchrotron radiolysis chemistry. Radiolyzed peptide products were detected within 10 ms of exposure to a white light synchrotron X-ray beam. Mass spectrometry techniques were used to characterize radiolytic modification to amino acids cysteine (Cys), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), proline (Pro), histidine (His), and leucine (Leu). A reactivity order of Cys, Met > Phe, Tyr, > Trp > Pro > His, Leu was determined under aerobic reaction conditions from MS/MS analysis of the radiolyzed peptide products. Radiolysis of peptides in 18O-labeled water under aerobic conditions revealed that oxygenated radical species from air and water both contribute to the modification of amino acid side chains. Cysteine and methionine side chains reacted with hydroxyl radicals generated from radiolysis of water as well as molecular oxygen. Phenylalanine and tyrosine residues were modified predominantly by hydroxyl radicals, and the source of modification of proline was exclusively through molecular oxygen.     

Dating silk by capillary electrophoresis mass spectrometry

A new capillary electrophoresis mass spectrometry (CE-MS) technique is introduced for age estimation of silk textiles based on amino acid racemization rates. With an L to D conversion half-life of ~2500 years for silk (B. mori) aspartic acid, the technique is capable of dating silk textiles ranging in age from several decades to a few-thousand-years-old. Analysis required only ~100 μg or less of silk fiber. Except for a 2 h acid hydrolysis at 110 °C, no other sample preparation is required. The CE-MS analysis takes ~20 min, consumes only nanoliters of the amino acid mixture, and provides both amino acid composition profiles and D/L ratios for ~11 amino acids.     

MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports

Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increase sample throughput, we have developed a novel in-gel digestion protocol where sample preparation is performed directly on a MALDI probe with prestructured sample support. The protocol consists of few sample-handling steps and has minimal consumption of reagents, making the protocol sensitive, timesaving, and cost-efficient. Performance of the on-probe sample preparation protocol was demonstrated by analysis of a set of rat liver proteins obtained from a fluorescently stained (Cy3 and SyproRuby) two-dimensional polyacrylamide gel. The success rate of protein identification by on-probe tryptic digestion and MALDI peptide mass mapping was 89%. The on-probe in-gel digestion procedure provided superior sensitivity and peptide mass mapping performance as compared to our standard in-gel digestion protocol. The on-probe digestion technique resulted in significantly improved amino acid sequence coverage of proteins, mainly due to efficient recovery and detection of large (>1.5 kDa) hydrophobic peptides. These observations indicate that numerous tryptic peptides are lost when using the standard in-gel digestion methods and sample preparation techniques for MALDI MS. This study also demonstrates that the on-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications.     

Impact of peptide modifications on the isobaric tags for relative and absolute quantitation method accuracy

In this study, the impact of amino acid modifications on the accuracy of the iTRAQ (isobaric tags for relative and absolute quantitation) method was evaluated. MCF-7 breast cancer cells, cultured in the presence of 17β-estradiol and tamoxifen, were used as a model system. The cells were labeled and analyzed by reversed-phase liquid chromatography and pulsed Q dissociation ion trap tandem mass spectrometry detection. Database searching was performed by using various combinations of amino acid modification allowances, i.e, Lys/Tyr/Cys and amino terminal iTRAQ labeling, Lys methylation, acetylation and carbamylation, and Cys/Met oxidation. Other than the intended Lys/amino terminal iTRAQ labeling, such modifications occur as a result of either enzymatic or sample preparation related reactions and are typically ignored in quantitation analysis to minimize the rate of false-positive peptide identifications. The study revealed that the modifications with the greatest impact on protein identification and quantitation pertain to Lys and Tyr amino acid residues, that by enabling such modifications the number and type of identified proteins will change (by up to 10%), and that the rate of false-positive protein identifications can be maintained below an upper threshold of 5% if appropriate data filtering conditions are used. In addition, the interference of possible posttranslational modifications (i.e., phosphorylation) with iTRAQ quantitation was examined.     

Comparison of fluorescence, laser-induced fluorescence, and ultraviolet absorbance detection for measuring HPLC fractionated protein/peptide mixtures

Proteomics is the study of all proteins in a biological sample. High-pressure liquid chromatography coupled online with mass spectrometry (HPLC/MS) is currently the method of choice for proteomic analysis. Proteins are extracted, separated at the protein or peptide level (after enzymatic digestion), and fractions are analyzed by HPLC/MS. Detection during off-line fractionation is generally conducted using UV-vis, which is not sensitive enough to distinguish fractions having the largest concentration of proteins/peptides and should not be combined prior to HPLC/MS. To overcome this deficiency, we utilize fluorescence or UV-laser induced fluorescence (UV-LIF) detection for measuring proteins/peptides during the off-line fractionation. Fluorescence detection allows low-abundance proteins/peptides that contain aromatic amino acids to be measured. In this study, peptide/protein samples fractionated using ion-exchange chromatography were detected using UV absorbance, fluorescence, and UV-LIF. The results indicated that fluorescence and UV-LIF were able to detect the lower abundance proteins/peptides to give a more representative chromatogram, allowing the analyst to decide which fractions should be combined prior to HPLC/tandem mass spectrometry (MS/MS) analysis.     

Attomole amino acid determination by capillary zone electrophoresis with thermooptical absorbance detection

Attomole quantities of 4-(dimethylamino)azobenzene-4'-sulfonyl chloride derivatized amino acids are separated by using capillary zone electrophoresis in a mixed acetonitrile/aqueous buffer system. Detection is performed with an on-column thermooptical absorbance detection technique based on a 130-mW argon ion pump laser. Detection limits for the concentration of analyte injected onto the column range from 5 x 10(-8) M for methionine to 5 x 10(-7) M for aspartic acid. Only 37 amol of methionine and 450 amol of aspartic acid are contained within the subnanoliter injection volume. It is interesting to note that these limits are a factor of 4 superior to the best fluorescence detection limit reported for chromatographic separation of amino acids. A subnanoliter sample of derivatized human urine was analyzed with this technique; quantities of amino acids contained within the sample are 3 orders of magnitude greater than the detection limit.     

Residue-specific mass signatures for the efficient detection of protein modifications by mass spectrometry

Currently available mass spectrometric (MS) techniques lack specificity in identifying protein modifications because molecular mass is the only parameter used to characterize these changes. Consequently, the suspected modified peptides are subjected to tandem MS/MS sequencing that may demand more time and sample. We report the use of stable isotope-enriched amino acids as residue-specific "mass signatures" for the rapid and sensitive detection of protein modifications directly from the peptide mass map (PMM) without enrichment of the modified peptides. These mass signatures are easily recognized through their characteristic spectral patterns and provide fingerprints for peptides containing the same content of specific amino acid residue(s) in a PMM. Without the need for tandem MS/MS sequencing, a peptide and its modified form(s) can readily be identified through their identical fingerprints, regardless of the nature of modifications. In this report, we demonstrate this strategy for the detection of methionine oxidation and protein phosphorylation. More interestingly, the phosphorylation of a histone protein, H2A.X, obtained from human skin fibroblast cells, was effectively identified in response to low-dose radiation. In general, this strategy of residue-specific mass tagging should be applicable to other posttranslational modifications.     

CE/electrospray ionization-MS analysis of underivatized d/l-amino acids and several small neurotransmitters at attomole levels through the use of 18-crown-6-tetracarboxylic acid as a complexation reagent/background electrolyte

A new capillary electrophoresis/mass spectrometry technique is introduced for attomole detection of primary amines (including several neurotransmitters), amino acids, and their d/l enantiomers in one run through the use of a complexation reagent while using only approximately 1 nL of sample. The technique uses underivatized amino acids in conjunction with an underivatized capillary, which significantly reduces cost and analysis time. It was found that when (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TCA, MW 440) was used as the background electrolyte/complexation reagent during the capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of underivatized amino acids, stable complexes were formed between the amino acids and the 18-C-6-TCA molecules. These complexes, which exhibited high ionization efficiencies, were detectable at attomole levels for most amino acids. The detection limits of the AA/18-C-6-TCA complexes were on the average more than 2 orders of magnitude lower than that of the free amino acids in solution. In addition to lower detection limits under CE/ESI-MS, a solution of 18-C-6-TCA in the concentration range of 5-30 mM provided high separation efficiency for mixtures of l-amino acids as well as mixtures of d/l-amino acids. By using a solution of 18-C-6-TCA as the background electrolyte in conjunction with an underivatized, 130-cm-long, 20-microm-i.d., 150-microm-o.d. fused-silica capillary and by monitoring the m/z range of the amino acid/18-C-6-TCA complexes (m/z 515-700), most of the standard amino acids and many of their enantiomers were separated and detected with high separation efficiency and high sensitivity (nanomolar concentration detection limits) in one run. The solutions of 18-C-6-TCA also worked well as the CE/ESI-MS BGE for low-level detection of several neurotransmitters and some of their d/l enantiomers as well as for the analysis of amino acids at endogenous levels in lysed red blood cells.     

Radiolytic modification of acidic amino acid residues in peptides: probes for examining protein-protein interactions

Hydroxyl radical-mediated footprinting coupled with mass spectroscopic analysis is a new technique for mapping protein surfaces, identifying structural changes modulated by protein-ligand binding, and mapping protein-ligand interfaces in solution. In this study, we examine the radiolytic oxidation of aspartic and glutamic acid residues to probe their potential use as structural probes in footprinting experiments. Model peptides containing Asp or Glu were irradiated using white light from a synchrotron X-ray source or a cesium-137 gamma-ray source. The radiolysis products were characterized by electrospray mass spectrometry including tandem mass spectrometry. Both Asp and Glu are susceptible to radiolytic oxidization by gamma-rays or synchrotron X-rays. Radiolysis results primarily in the oxidative decarboxylation of the side chain carboxyl group and formation of an aldehyde group at the carbon next to the original carboxyl group, giving rise to a characteristic product with a -30 Da mass change. A similar oxidative decarboxylation also takes place for amino acids with C-terminal carboxyl groups. The methylene groups in the Asp and Glu side chains also undergo oxygen addition forming ketone or alcohol groups with mass changes of +14 and +16 Da, respectively. Characterizing the oxidation reactions of these two acidic residues extends the number of useful side chain probes for hydroxyl radical-mediated protein footprinting from 10 (Cys, Met, Trp, Tyr, Phe, Arg, Leu, Pro, His, Lys) to 12 amino acid residues, thus enhancing our ability to map protein surface structure and in combination with previously identified basic amino acid probes can be used to examine molecular details of protein-protein interactions that are driven by electrostatics.     

Capillary electrophoresis-fourier transform ion cyclotron resonance mass spectrometry for the identification of cationic metabolites via a pH-mediated stacking-transient isotachophoretic method

Capillary electrophoresis-mass spectrometry (CE-MS) is still widely regarded as an emerging tool in the field of metabolomics and metabolite profiling. A major reason for this is a reported lack of sensitivity of CE-MS when compared to gas chromatography-mass spectrometry GC/MS and liquid chromatography-mass spectrometry. The problems caused by the lack of sensitivity are exacerbated when CE is coupled to Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), due to the relatively low data acquisition rate of FT-ICR MS. Here, we demonstrate the use of an online CE sample preconcentration method that uses a combination of pH-mediated stacking and transient isotachophoresis, coupled with FT-ICR MS to improve the overall detection of cationic metabolites in the bacterium Desulfovibrio vulgaris Hildenborough. This method showed a significant increase in signal-to-noise ratio when compared to CE normal sample stacking, while providing good separation efficiency, reproducibility, and linearity. Detection limits for selected amino acids were between 0.1 and 2 microM. Furthermore, FT-ICR MS detection consistently demonstrated good mass resolution and sub-ppm mass accuracy.     

Detection of functional ricin by immunoaffinity and liquid chromatography-tandem mass spectrometry

The toxin ricin is a biological weapon that may be used for bioterrorist purposes. As a member of the group of ribosome-inactivating proteins (RIPs), ricin has an A-chain possessing N-glycosidase activity which irreversibly inhibits protein synthesis. In this paper, we demonstrate that provided appropriate sample preparation is used, this enzymatic activity can be exploited for functional ricin detection with sensitivity similar to the best ELISA and specificity allowing application to environmental samples. Ricin is first captured by a monoclonal antibody directed against the B chain and immobilized on magnetic beads. Detection is then realized by determination of the adenine released by the A chain from an RNA template using liquid chromatography coupled to tandem mass spectrometry. The immunoaffinity step combined with the enzymatic activity detection leads to a specific assay for the entire functional ricin with a lower limit of detection of 0.1 ng/mL (1.56 pM) after concentration of the toxin from a 500 microL sample size. The variability of the assay was 10%. Finally, the method was applied successfully to milk and tap or bottled water samples.     

Terbutaline enantiomer separation and quantification by complexation and field asymmetric ion mobility spectrometry-tandem mass spectrometry

Recently, we introduced a new approach to chiral separation and analysis of amino acids by chiral complexation and electrospray high-field asymmetric waveform ion mobility spectrometry coupled to mass spectrometry (ESI-FAIMS-MS). In the present work, we extended this approach to the separation of the drug compound terbutaline. Terbutaline enantiomers were complexed with metal ions and an amino acid to form diastereomeric complexes of the type [M(II)(L-Ref)2((+)/(-)-A)-H](+), where M(II) is a divalent metal ion, L-Ref is an amino acid in its L-form, and A is the terbutaline analyte. When metal and reference compound were suitably chosen, these complexes were separable by FAIMS. We also detected and characterized larger clusters that were transmitted at distinct FAIMS compensation voltages (CV), disturbing data analysis by disintegrating after the FAIMS separation and forming complexes of the same composition [M(II)(L-Ref)2((+)/(-)-A)-H](+), thus giving rise to additional peaks in the FAIMS CV spectra. This undesired phenomenon could be largely avoided by adjusting the mass spectrometer skimmer voltages in such a way that said larger clusters remained intact. In the quantitative part of the present work, we achieved a limit of detection of 0.10% (-)-terbutaline in a sample of (+)-terbutaline. The limit of detection and analysis time per sample compared favorably to literature values for chiral terbutaline separation by HPLC and CE.     

Comparison of high-field asymmetric waveform ion mobility spectrometry with GC methods in analysis of haloacetic acids in drinking water

Haloacetic acids (HAAs) are major byproducts of chlorination of drinking water. Electrospray ionization high-field asymmetric waveform ion mobility spectrometry mass spectrometry (ESI-FAIMS-MS) provides a tool for direct monitoring of these compounds. However, treated drinking water samples can be challenging to analyze due to the large number of chemicals present and due to matrix effects that can hinder quantitation of analytes. We developed a standard addition ESI-FAIMS-MS method that permits submicrogram per liter detection of haloacetic acids and overcomes matrix effects. An advantage of FAIMS is increased selectivity through a significant reduction in the chemical background from ESI. Moreover, detection limits with this method are much lower than with previously existing GC and GC/MS methods, and quantitation results compare favorably with other existing methods. This new method does not require sample preparation or chromatographic separation and provides a fast, simple, sensitive, and selective method for monitoring HAAs.     

Complications in the assignment of 14 and 28 Da mass shift detected by mass spectrometry as in vivo methylation from endogenous proteins

Identification of protein methylation sites typically starts with database searching of MS/MS spectra of proteolytic digest of the target protein by allowing addition of 14 and 28 Da in the selected amino acid residues that can be methylated. Despite the progress in our understanding of lysine and arginine methylation, substrates and functions of protein methylation at other amino acid residues remain unknown. Here we report the analysis of protein methylation for p53, SMC3, iNOS, and MeCP2. We found that a large number of peptides can be modified on the lysine, arginine, histidine, and glutamic acid residues with a mass increase of 14 or 28 Da, consistent with methylation. Surprisingly, a majority of which did not demonstrate a corresponding mass shift when cells were cultured with isotope-labeled methionine, a precursor for the synthesis of S-adenosyl-l-methionine (SAM), which is the most commonly used methyl donor for protein methylation. These results suggest the possibility of either exogenous protein methylation during sample handling and processing for mass spectrometry or the existence of SAM-independent pathways for protein methylation. Our study found a high occurrence of protein methylation from SDS-PAGE isolated endogenous proteins and identified complications for assigning such modifications as in vivo methylation. This study provides a cautionary note for solely relying on mass shift for mass spectrometric identification of protein methylation and highlights the importance of in vivo isotope labeling as a necessary validation method.     

Validated quantitation of underivatized amino acids in human blood samples by volatile ion-pair reversed-phase liquid chromatography coupled to isotope dilution tandem mass spectrometry

Quantitation of amino acids in complex matrixes without derivatization is advantageous; however, difficulties exist in both the separation and the detection of those compounds. A validated method that is based on the use of volatile ion-pair liquid chromatography coupled to stable isotope dilution tandem mass spectrometry has been developed for the simple and accurate quantitation of underivatized amino acids in biological samples. Sufficient separation of 22 underivatized amino acids was achieved on a C18 column in 36 min using perfluoroheptanoic acid (PFHA) and trifluoroacetic acid (TFA) as mobile phase modifiers. The collisionally activated dissociation spectra of the amino acids were investigated and the transitions of [M + H]+ --> [M + H - 46]+, which are specific to alpha-amino acids, were used for the detection of most amino acids and their stable isotopes. The calibration curves were linear over the range of 0.10-100 microg/mL, and the detection limits were 0.03-20 pmol on column. The quantitative results by this method were compared with those by an established OPA-derivatization HPLC method in the assay of 8 human serum samples, and better recovery and precision data of this method were observed. The method was also applied to the neonatal screening for phenylketonuria (PKU) with dry blood spots, and the results were satisfactory. This is the first time that all proteinogenic amino acids have been quantified directly from biological extracts without any kind of derivaization. The technique shows potential for routine determination of amino acids and analogous compounds in complex matrixes.     

Analysis of underivatized amino acids and their D/L-enantiomers by sheathless capillary electrophoresis/electrospray ionization mass spectrometry

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of underivatized amino acids and the separation of their D/L-enantiomers. Under full-scan mode, all standard protein amino acids were separated and detected at low-femtomole levels using a 130-cm-long, 20-microm-i.d., 150-microm-o.d. underivatized fused-silica capillary with 1 M formic acid as the background electrolyte. The CE/ESI-MS technique was also applied to the separation of L-arginine from L-canavanine (a close analogue of arginine where the terminal methylene linked to the guanidine group of arginine is replaced by an oxygen atom) in a complex mixture containing all standard protein amino acids. The utility of CE/ESI-MS in the analysis of real-world samples was demonstrated by the identification of two metabolic diseases (PKU and tyrosinemia) through blood analysis with minimal sample preparation. In addition, the on-line separation of 11 underivatized L-amino acids from their D-enantiomers was achieved by using a 30 mM solution of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as the background electrolyte.     

Determination of methionine and selenomethionine in yeast by species-specific isotope dilution GC/MS

A method for the simultaneous determination of methionine (Met) and selenomethionine (SeMet) in yeast using species-specific isotope dilution (ID) gas chromatography/mass spectrometry (GC/MS) is described. Samples were digested by refluxing for 16 h with 4 M methanesulfonic acid. Analytes were derivatized with methyl chloroformate and extracted into chloroform for GC/MS analysis. In addition to use of commercially available 13C-enriched Met and SeMet spikes for species specific ID analysis, a 74Se-enriched SeMet spike was also available for comparison of results. In selective ion monitoring mode, the intensities of ions at m/z 221, 222, 269, 270, and 263 were used to calculate the 221/222, 269/270, and 269/263 ion ratios for quantification of Met and SeMet. Concentrations of 5959 +/- 33 and 3404 +/- 12 microg g(-1) (one standard deviation, n = 6) with relative standard deviations of 0.55 and 0.36% for Met and SeMet, respectively, were obtained using 13C-enriched spikes. A concentration of 3417 +/- 8 microg g(-1) (one standard deviation, n = 6) was obtained using the 74Se-enriched SeMet spike. The concentration of SeMet measured in the yeast is equivalent to 66.43 +/- 0.24% of total Se and 30.31 +/- 0.11% of total Met is in the form of SeMet. Method detection limits (three times the standard deviation) of 3.4 and 1.0 microg g(-1) were estimated for Met and SeMet, respectively, based on a 0.25-g subsample of yeast with 1 mL of extract used for derivatization. A similar concentration of 5930 +/- 29 microg g(-1) (one standard deviation, n = 4) for Met and a lower concentration of 2787 +/- 49 microg g(-1) (one standard deviation, n = 4) for SeMet were obtained for this yeast sample using species-specific ID analysis based on GC/MS with 13C-enriched Met and SeMet spikes when a 2-h open microwave digestion approach using 8 M methanesulfonic acid was used.     

Development of a methodology based on metal-catalyzed oxidation reactions and mass spectrometry to determine the metal binding sites in copper metalloproteins

Efforts have been made to develop a method that uses metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) to identify the binding site of copper in metalloproteins. This method uses MCO reactions to oxidize the amino acids in the metal-binding site and MS to identify the amino acids that have been oxidized. Several reaction conditions, including Cu(II)/ascorbate/O2, Cu(II)/O2/H2O2, and Cu(II)/ascorbate/O2/H2O2, have been tested at varying concentrations to find the optimum conditions for specific oxidation of only the amino acids bound to copper. For small peptides, such as angiotensin I (Agt I) and [Gln11]-amyloid-beta-protein fragment 1-16 (A beta(1-16)), the optimum conditions for specific modification involve the use of Cu(II)/ascorbate/O2. For a larger protein, azurin, the speed and specificity of the MCO reactions are enhanced by the presence of a relatively high concentration of ascorbate (100 mM) and a small concentration of H2O2 (1 mM). Optimized reaction conditions combined with MS/MS and MSn analysis on a quadrupole ion trap mass spectrometer allow the copper-binding sites to be specifically identified. For Agt I and A beta(1-16), the amino acids bound to copper can be identified without any false positives. For azurin, four of the five amino acids bound to copper are identified with one false positive. This false positive, however, corresponds to the oxidation of Met44, which is probably due to its susceptibility to oxidation and its proximity to the only residue not identified (i.e., Gly45). The results altogether suggest that MCO reactions and MS provide a very promising approach for identifying the amino acid residues bound to copper in metalloproteins.     

Automated 96-well SPE and LC-MS-MS for determination of protease inhibitors in plasma and cartilage tissues

Bioanalytical methods based on automated solid-phase extraction (SPE) and high-performance liquid chromatography with electrospray tandem mass spectrometry (LC-MS-MS) have been developed and utilized for the determination of MMP inhibitors in plasma and cartilage tissues. The SPE methods were automated using a 96-well extraction plate and a 96-channel programmable liquid-handling workstation. The LC-MS-MS methods were developed using a rapid gradient LC separation, followed by sample introduction through an ionspray interface in the positive ion mode and tandem mass spectrometric detection with selected reaction monitoring. In the optimized SPE methods, crude plasma or ground cartilage supernatant samples were loaded onto an SPE plate to remove proteins and other interfering components in the matrixes to render relatively clean extracts for LC-MS-MS analysis. Compared to the simple plasma protein precipitation method, the automated SPE method afforded significant time-saving in sample preparation and improved sensitivity in MS detection. The methods were validated and successfully applied to the analysis of protease inhibitors in plasma and cartilage tissues.