High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins

We have developed a method for intact mass analysis of detergent-solubilized and purified integral membrane proteins using liquid chromatography–mass spectrometry (LC–MS) with methanol as the organic mobile phase. Membrane proteins and detergents are separated chromatographically during the isocratic stage of the gradient profile from a 150-mm C3 reversed-phase column. The mass accuracy is comparable to standard methods employed for soluble proteins; the sensitivity is 10-fold lower, requiring 0.2–5 μg of protein. The method is also compatible with our standard LC–MS method used for intact mass analysis of soluble proteins and may therefore be applied on a multiuser instrument or in a high-throughput environment.     

Quantification of corticosteroids in bovine urine using selective solid phase extraction and reversed-phase liquid chromatography/tandem mass spectrometry

This paper presents the development of a simple liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to determine corticosteroids in bovine urine sample matrices. This method uses a single phase extraction (SPE) for cleaning of the sample with an Oasis MAX cartridge at pH 9.0–9.5 and elution by a neutral organic solvent (acetonitrile/dichloromethane), followed by separation on a GEMINI C18 column in the gradient mode with acetate buffer (pH 4.1)/methanol. A triple quadrupole mass spectrometer equipped with a multimode ion source, set to negative atmospheric pressure chemical ionization (APCI) in the multiple reaction monitoring mode was used for detection. The main advantage of this method over other commonly used methods includes the use of SPE with a low volume cartridge for sample preparation and no ion suppression effects from matrix components of the urine samples in the LC–MS/MS analysis. This allowed a reduction the quantification limits (decision limits, CCα) for the first time to 0.1 μg/L (1 and 0.2 μg/L for triamcinolone and flumethasone, respectively). The developed method was validated in accordance with the European Union Commission Decision 2002/657 EC. The recoveries and within-laboratory reproducibility varied from 77% to 115% and 87% to 107.5%, respectively, at 2, 3, and 4 μg/L levels of corticosteroids. The relative standard deviation (RSD) of the measurements was lower than 30%. The decision limit was calculated by multiplying the signal-to-noise ratio by 3 and the obtained values were in the range of 0.1–1.0 μg/L, confirmed by the analysis of twenty blank samples, which were spiked at the desired concentrations. The detection capability was calculated by the addition of the decision limit and the standard deviation followed by multiplication by 1.64 of the within-laboratory reproducibility at 2 μg/L of corticosteroids. The method was applied to four urine samples, giving concentrations of prednisolone (PRED) residues in the range from 0.3 to 0.9 μg/L.     

Optimization of digestion parameters for protein quantification

We present a rapid and efficient in-solution enzymatic digestion protocol suitable for mass spectrometry-based absolute protein quantification techniques. The digestion method employs RapiGest SF (an acid-labile surfactant), an excess amount of modified trypsin (enzyme-to-substrate ratio of 2.5:1), and an incubation time of 2 h. No reduction/alkylation reagents are used. Digestion parameters were varied systematically to monitor their effect on rate and completeness of digestion. To demonstrate the general applicability of the method, the optimization was done using a viral hemagglutinin (HA) as a model protein and then applied to ricin, a potent protein toxin extracted from the castor bean (Ricinus communis). The parameters that were optimized included incubation time, concentration of RapiGest SF, enzyme-to-substrate ratio, and incubation temperature. The optimization was done by comparing the yields from two protein-specific peptides originating from two different sites of the HA protein. The analysis was performed by liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode using isotopically labeled peptide standards for quantification.     

Minimizing artifactual elevation of lipid peroxidation products (F2-isoprostanes) in plasma during collection and storage

F₂-isoprostanes (F₂-IsoP’s) are reliable measures of in vivo lipid oxidation, but care is required to prevent artifactual elevation. We examined the effects of blood collection and storage on plasma F₂-IsoP’s. Blood was collected into EDTA/butylated hydroxytoluene/reduced glutathione (EDTA/BHT/GSH) or EDTA, at 4 °C or room temperature. Plasma was stored at −20 or −80 °C for 1 or 6 months before F₂-IsoP’s were assayed by GC–MS. The temperature of blood collection did not affect F₂-IsoP’s. However, storage at −20 °C or collection into EDTA resulted in significant increases in F₂-IsoP’s. Blood collection into EDTA/BHT/GSH and storage at −80 °C minimizes artifactual elevation of plasma F₂-IsoP’s.     

SMART Digest™ compared with pellet digestion for analysis of human immunoglobulin G1 in rat serum by liquid chromatography tandem mass spectrometry

The newly developed SMART Digest™ kit was applied for the sample preparation of human immunoglobulin G1 (hIgG1) in rat serum prior to qualitative and quantitative analyses by liquid chromatography tandem mass spectrometry (LC–MS/MS). The sequence coverages obtained for the light and heavy chains of hIgG1A were 50 and 76%, respectively. The calibration curve was linear from 1.00 to 1000 μg/ml for three of four generic peptides. Overall, the SMART Digest™ kit resulted in similar quantitative data (linearity, sensitivity, accuracy, and precision) compared with the pellet digestion protocol. However, the SMART Digest™ required only 2 h of sample preparation with fewer reagents.     

An in-gel digestion procedure that facilitates the identification of highly hydrophobic proteins by electrospray ionization-mass spectrometry analysis

A procedure is described for in-gel tryptic digestion of proteins that allows the direct analysis of eluted peptides in electrospray ionization (ESI) mass spectrometers without the need of a postdigestion desalting step. It is based on the following principles: (a) a thorough desalting of the protein in-gel before digestion that takes advantage of the excellent properties of acrylamide polymers for size exclusion separations, (b) exploiting the activity of trypsin in water, in the absence of inorganic buffers, and (c) a procedure for peptide extraction using solvents of proven efficacy with highly hydrophobic peptides. Quality of spectra and sequence coverage are equivalent to those obtained after digestion in ammonium bicarbonate for hydrophilic proteins detected with Coomassie blue, mass spectrometry-compatible silver or imidazole-zinc but are significantly superior for highly hydrophobic proteins, such as membrane proteins with several transmembrane domains. ATPase subunit 9 (GRAVY 1.446) is a membrane protein channel, lipid-binding protein for which both the conventional in-gel digestion protocol and in solution digestion failed. It was identified with very high sequence coverage. Sample handling after digestion is notably simplified as peptides are directly loaded into the ESI source without postdigestion processing, increasing the chances for the identification of hydrophobic peptides.     

Gas chromatography–mass spectrometry-based simultaneous quantitative analytical method for urinary oxysterols and bile acids in rats

A simultaneous quantitative assay method for urinary oxysterols and bile acids using GC–MS was developed to investigate the mechanism of liver toxicity induced by drugs or chemicals. Sample preparations were optimized by exploring various extraction solvents, derivatization reagents, and hydrolysis methods to achieve reliable and maximum sensitivity for these two different compound classes. As a result, satisfactory accuracy, precision, and sensitivity were obtained in the validation. The method was then applied to quantify urinary oxysterols and bile acids produced from liver toxicity induced by atorvastatin (250 mg/kg/day). From the results, increases in bile acid levels and decreases in the concentration ratio between cholic acid and chenodeoxycholic acid, which are the distinguishing phenomena observed in serum or bile for liver toxicity, were also observed in urine. Additionally, the mechanism of liver toxicity was investigated with the urinary concentration ratio of product to precursor in the metabolic pathway from cholesterol to bile acids. The results indicated that enzyme activities related to the production and degradation of bile acids, not oxysterols, were significantly changed from liver toxicity. Thus, it was concluded that urinary levels of oxysterols and bile acids could be useful tools for checking liver toxicity and investigating its mechanism.     

Lipid mapping of colonic mucosa by cluster TOF-SIMS imaging and multivariate analysis in cftr knockout mice

The cftr knockout mouse model of cystic fibrosis (CF) shows intestinal obstruction; malabsorption and inflammation; and a fatty acid imbalance in intestinal mucosa. We performed a lipid mapping of colon sections from CF and control (WT) mice by cluster time of flight secondary-ion mass spectrometry (TOF-SIMS) imaging to localize lipid alterations. Data were processed either manually or by multivariate statistical methods. TOF-SIMS analysis showed a particular localization for cholesteryl sulfate at the epithelial border, C16:1 fatty acid in Lieberkühn glands, and C18:0 fatty acid in lamina propria and submucosa. Significant increases in vitamin E (vE) and C16:0 fatty acid in the epithelial border of CF colon were detected. Principal component analysis (PCA) and partitioning clustering allowed us to characterize different structural regions of colonic mucosa according to variations in C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C20:3, C20:4, and C22:6 fatty acids; phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol glycerolipids; cholesterol; vitamin E; and cholesteryl sulfate. PCA on spectra from Lieberkühn glands led to separation of CF and WT individuals. This study shows for the first time the spatial distribution of lipids in colonic mucosa and suggests TOF-SIMS plus multivariate analyses as a powerful tool to investigate disease-related tissue spatial lipid signatures.     

Source separation,selective collection and in reactor digestion of biowaste

Biowaste or the organic fraction of domestic waste, for instance kitchen, fruit and garden waste, is collected selectively in several European communities. The complementary fraction is called the dry or non recyclable fraction. A Dutch study reported that 92% of the participants that have a weekly collection service of both fractions (biowaste and non recyclable fraction) and 80% of the participants in the alternating collection program (one week biowaste and the next week non recyclable fraction) are pleased with separate collection of biowaste. Dominating problems that arise in case of alternating collection are a repulsive odor and an infestation with flies and maggots. By expanding the definition of biowaste to include non recyclable or soiled paper like dirty newspapers, table napkins and paper handkerchiefs, most of these problems can be overcome without changing the way compostable waste is collected and processed. The expanded definition of biowaste was used in this paper. Over a 12 month period a quality survey of the collected biowaste was conducted by the composting facility Intercompost, Hoeselt, Belgium. A special aspect was the fact that in one participating community baby diapers were included in the soiled paper fraction; this is called biowaste+. The biowaste+ had a 10% non recyclable paper fraction opposed to only 1–2% of non recyclable paper present in the conventional biowaste. Baby diapers were a rather notable part (more than 80%) of this non recyclable paper fraction of biowaste+ and as a consequence might contribute to a large extent to improve the collection and treatment of biowaste. It was demonstrated that rural districts yielded about 35% more biowaste than more urban districts; resp. ± 122 kg biowaste/capita.year versus ±90 kg biowaste/capita.year. In Hoeselt the biowaste+ yield was about 130 kg/capita.year. Biowaste+ is also separately collected in another Belgium community, namely Brecht. The purity level of the biowaste+ and the amount of non recyclable paper (including diapers) were comparable with the results of Hoeselt. In Hoeselt the biowaste+ fraction is composted aerobically. In Brecht on the other hand, the biowaste+ is processed using the Dry Anaerobic Composting process (DRANCO). The latter process is discussed in more detail. The biological start-up of the dry anaerobic composting installation at Brecht, Belgium, is reported. The reactor has a total volume of 808 m³ and a design capacity of 730 m³. After 2 months of start up, the fermentor was at full loading rate, i.e. 8 kg bVS/m³ _reactor .day and the installation was working at full capacity, i.e. 40 ton/day. After 1 year of full-scale operation, the installation can handle peak loads of 60 ton/day. The average biogas production is 107 m³/ton with an average methane content of 55%. This corresponds to an annual gross energy production of approximately 600 toe (ton oil equivalent). The average biogas production rate is 4.0 m³/m³ _reactor .day. The end product of the aerobic process in Hoeselt and the end product in Brecht, the Humotex, are of similar composition. Both are of high quality and are sold as a soil conditioner or as a potting substrate. However, the double processed compost has a garanteed hygienic quality which makes it a product suitable for export.     

Genome-wide analysis of terpene synthases in soybean: functional characterization of GmTPS3

Terpenes (terpenoids or isoprenoids) constitute a large class of plant natural products and play numerous functional roles in primary and secondary metabolism as well as inecological interactions. This study presents a genomic analysis of 23 putative soybean (Glycine max) terpene synthase genes (GmTPSs) distributed over 10 of 20 chromosomes. The GmTPSs are grouped into six types based on gene architecture and sequence identity. Sequence alignment indicates that most GmTPSs contain the conserved aspartate-rich DDX₂D motif, and two clades encoded by TPS-a and TPS-b contain variations of an arginine-rich RRX₈W motif. Quantitative real-time PCR analysis demonstrated that GmTPSs were predominantly expressed in reproductive organs. Heterologous expression followed by enzymatic assay suggested that GmTPS3 functions as a geraniol synthase. We also generated transgenic tobacco plants ectopically expressing GmTPS3. In dual-choice feeding-preference and force-feeding assays, the transgenic tobacco lines expressing GmTPS3 exhibited enhanced resistance to cotton leafworms and an increased level of geraniol. Taken together, these data provide a comprehensive understanding of the TPS family in soybeans and suggest a promising approach to engineering transgenic plants with enhanced insect resistance.     

Structural analysis of arabinoxylans isolated from ball-milled switchgrass biomass

Ball-milled alcohol-insoluble residue (AIR) was prepared from switchgrass (Panicum virgatum var Alamo) and sequentially extracted with 50 mM ammonium oxalate buffer, 50 mM sodium carbonate, 1 M KOH containing 1% NaBH₄, and 4 M KOH containing 1% NaBH₄. Arabinoxylan was the most abundant component of the 1 M KOH-extracted fraction, which was treated with endoxylanase to generate oligosaccharides. Gel-permeation chromatography of these oligosaccharides produced three size-homogeneous oligosaccharide fractions with molecular weights of 678, 810, and 1074 Da, corresponding to 5, 6, and 8 pentose units, respectively. Detailed structural analysis of these oligosaccharides was performed using methylation analysis, multiple-step mass spectrometry (ESIMSⁿ), and 1D and 2D NMR spectroscopy. The preferred gas-phase fragmentation pathways were identified for these oligosaccharides, providing extensive sequence information that was completely consistent with structures determined by ab initio NMR analysis. These results demonstrate the high information content of ESIMSⁿ analysis when applied to cell-wall-derived oligosaccharides and provide standard data that will facilitate the analysis of cell-wall polysaccharide fragments with a sensitivity that is sufficient for the analysis of samples obtained from dissected tissues as well as other small samples.     

Four novel GALC gene mutations in two Chinese patients with Krabbe disease

Krabbe disease (OMIM #245200) is a rare autosomal recessive leukodystrophy caused by deficiency of galactocerebrosidase (GALC) activity. We identified four novel mutations of the GALC gene in two unrelated Chinese families with Krabbe disease: one insertion mutation, c.1836_1837insT, and one nonsense mutation, c.599C>A (p.S200X), in an infantile patient, and one deletion mutation, c.1911+1_1911+5delGTAAG, and one missense mutation, c.2041G>A, in an adult late-onset patient. This is the first identification of GALC mutations in the Chinese population.     

Method for determination of methadone in exhaled breath collected from subjects undergoing methadone maintenance treatment

At present drugs of abuse testing using exhaled breath as specimen is only possible for alcohol. However, we recently discovered that using modern liquid chromatography–mass spectrometry technique amphetamine and methamphetamine is detectable in exhaled breath following intake in drug addicts. We therefore undertook to develop a method for determination of methadone in exhaled breath from patients undergoing methadone maintenance treatment. Exhaled breath was collected from 13 patients after intake of the daily methadone dose. The compounds were trapped by filtering the air through a C18 modified silica surface. After elution of any trapped methadone the extract was analysed by a combined liquid chromatography–tandem mass spectrometry method. Recovery of trapped methadone from the filter surface was 96%, no significant matrix effect was observed, and the quantification using methadone-d3 as an internal standard was accurate (<10% bias) and precise (coefficient of variation 1.6–2.0%). Methadone was indisputably identified by means of the mass spectrometry technique in exhaled breath samples from all 13 patients. Identification was based on monitoring two product ions in selected reaction monitoring mode with correct relative ratio (±20%) and correct retention time. Excretion rates ranged from 0.39 to 78 ng/min. No methadone was detected in 10 control subjects. This finding confirms that breath testing is a new possibility for drugs of abuse testing. Collection of exhaled breath specimen is likely to be more convenient and safe as compared to other matrices presently in use.     

Scent chemistry and patterns of thermogenesis in male and female cones of the African cycad Encephalartos natalensis (Zamiaceae)

Most, if not all, extant cycads are pollinated by insects which use the cones as larval brood sites. These interactions appear to be mediated by cone volatiles, and, in some species, by patterns of thermogenesis. We investigated the chemical composition of volatile emissions and patterns of thermogenesis in cones of the South African cycad Encephalartos natalensis, using a gas chromatograph–mass spectrometer (GC–MS) and miniature temperature data loggers (ibuttons), respectively. This was done during various developmental stages (before and during receptivity and pollen release) for both female and male cones. A total of 31 compounds were identified in headspace samples; 17 of which were common to both sexes, 12 found only in male cones, and two found only in female cones. The major volatiles in pollen and female cones are (3E)-1,3-octadiene (averaging 54.25% and 15.82% of total emissions), (3E,5Z)-1,3,5-octatriene (averaging 13.37% and 47.66%), and α-pinene (averaging 16.29% and 12.24%). Female cones were not thermogenic before and during receptivity whereas pollen cones were thermogenic during pollen shedding. Thermogenesis of male cones occurred between 1400 h and 1530 h on successive afternoons, reaching an average of c. 10.5 °C above ambient temperature. Volatile emissions and thermogenesis occurred in association with insect activity on the cones suggesting that they both play a role in regulating insect behaviour.     

Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1′-hydroxymidazolam,and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry

Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC–MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1′-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D₅) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 μL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 μL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm × 100 mm, 3.5 μm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100–250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85–115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam.     

On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format

Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of analytical technologies for their characterization. However, incompatibility between separation and subsequent detection is often encountered. Here we demonstrate the utility of a generic on-line liquid chromatography–mass spectrometry (LC-MS) method operated in a two-dimensional format toward the rapid characterization of MAb charge and size variants. Using a single chromatographic system capable of running two independent gradients, up to six fractions of interest from an ion exchange (IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto a series of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysis of poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis with minimal sample handling. This technology provides rapid data turnaround time, a much needed feature during product characterization and development of multiple biotherapeutic proteins.     

Mass spectrometric analysis of rabbit and bovine trypsin-solubilized cytochrome b5

The sequence and blocking group of the amino-terminal 15 amino acids of rabbit trypsin-solubilized cytochrome b₅ were determined by liquid secondary ion mass spectrometry (LSIMS) and tandem mass spectrometry (MS/MS). The molecular weights of peptides generated from aStaphylococcus aureus V8 protease digest of this protein were determined by LSIMS analysis and the two peptides containing the blocked amino-terminus were sequenced by tandem mass spectrometry to yield the sequence; N-acetyl-Ala-Ala-Glu-Ser-Asp-Lys-Asp-Val-Lys-Tyr-Tyr-Thr-Leu-Glu-Glu. Comparison of this sequence with a recently reported cDNA sequence (Dariushet al., 1988) indicates that Gln at position 3 is selectively deamidated, although no other discrepancies were found. Intact rabbit and bovine trypsin-solubilized cytochrome b₅ were also analyzed by LSIMS on a high-field mass spectrometer equipped with a diode array detector. Mass measurement of the unresolved protonated molecular ion peak tops gave average molecular weights of 9462.2±2 and 9502.3±2 for bovine and rabbit trypsin-solubilized cytochrome b₅, respectively. In both cases, these molecular weights correspond to a cytochrome b₅ fragment consisting of amino acids Asp(7)-Arg(88). The average molecular weight for the rabbit amino-terminal-blocked form of trypsin-solubilized cytochrome b₅ was found to be 10,144.5±2, which was consistent with the molecular weight predicted for the extended N-acetylated form (residues 1–88) of M_r 10,146.1.     

Mouse liver NAD(P)H:quinone acceptor oxidoreductase: protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression in Escherichia coli, and enzyme activity analysis.

The amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) has been determined by tandem mass spectrometry and deduced from the nucleotide sequence of the cDNA encoding for the enzyme. The electrospray mass spectral analyses revealed, as previously reported (Prochaska HJ, Talalay P, 1986, J Biol Chem 261:1372-1378), that the 2 forms--the hydrophilic and hydrophobic forms--of the mouse liver quinone reductase have the same molecular weight. No amino acid sequence differences were found by tandem mass spectral analyses of tryptic peptides of the 2 forms. Moreover, the amino-termini of the mouse enzymes are acetylated as determined by tandem mass spectrometry. Further, only 1 cDNA species encoding for the quinone reductase was found. These results suggest that the 2 forms of the mouse quinone reductase have the same primary sequences, and that any difference between the 2 forms may be attributed to a labile posttranslational modification. Analysis of the mouse quinone reductase cDNA revealed that the enzyme is 273 amino acids long and has a sequence homologous to those of rat and human quinone reductases. In this study, the mouse quinone reductase cDNA was also ligated into a prokaryotic expression plasmid pKK233.2, and the constructed plasmid was used to transform Escherichia coli strain JM109. The E. coli-expressed mouse quinone reductase was purified and characterized. Although mouse quinone reductase has an amino acid sequence similar to those of the rat and human enzymes, the mouse enzyme has a higher NAD(P)H-menadione reductase activity and is less sensitive to flavones and dicoumarol, 2 known inhibitors of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteomic analysis of two functional states of the Golgi complex in mammary epithelial cells

Organellar compartments involved in secretion are expanded during the transition from late pregnancy (basal secretory state) to lactation (maximal secretory state) to accommodate for the increased secretory function required for copious milk production in mammary epithelial cells. The Golgi complex is a major organelle of the secretory pathway and functions to sort, package, distribute, and post-translationally modify newly synthesized proteins and membrane lipids. These complex functions of the Golgi are reflected in the protein complement of the organelle. Therefore, using proteomics, the protein complements of Golgi fractions isolated at two functional states (basal and maximal) were compared to identify some of the molecular changes that occur during this transition. This global analysis has revealed that only a subset of the total proteins is up-regulated from steady state during the transition. Identification of these proteins by tandem mass spectrometry has revealed several classes of proteins involved in the regulation of membrane fusion and secretion. This first installment of the functional proteomic analysis of the Golgi complex begins to define the molecular basis for the transition from basal to maximal secretion.     

Sequence analysis of peptide mixtures by automated integration of Edman and mass spectrometric data.

A computer algorithm is described that utilizes both Edman and mass spectrometric data for simultaneous determination of the amino acid sequences of several peptides in a mixture. Gas phase sequencing of a peptide mixture results in a list of observed amino acids for each cycle of Edman degradation, which by itself may not be informative and typically requires reanalysis following additional chromatographic steps. Tandem mass spectrometry, on the other hand, has a proven ability to analyze sequences of peptides present in mixtures. However, mass spectrometric data may lack a complete set of sequence-defining fragment ions, so that more than one possible sequence may account for the observed fragment ions. A combination of the two types of data reduces the ambiguity inherent in each. The algorithm first utilizes the Edman data to determine all hypothetical sequences with a calculated mass equal to the observed mass of one of the peptides present in the mixture. These sequences are then assigned figures of merit according to how well each of them accounts for the fragment ions in the tandem mass spectrum of that peptide. The program was tested on tryptic and chymotryptic peptides from hen lysozyme, and the results are compared with those of another computer program that uses only mass spectral data for peptide sequencing. In order to assess the utility of this method the program is tested using simulated mixtures of varying complexity and tandem mass spectra of varying quality.