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Sullivan B, Addona TA, Carr SA. Selective detection of glycopeptides on ion trap mass spectrometers. Analytical Chemistry 76;2004:3112–3118. [PubMed]
Optimized conditions for generating carbohydrate-specific marker ions are presented to detect glycopeptides in glycoprotein digests. In-source collision-induced dissociation is used to generate ions of m/z 204 (HexNAc+), 292 (NeuAc+), and 366 (Hex-HexNAc+). Source conditions are optimized for the Finnigan LCQ-Deca 3D ion-trap mass spectrometer, but the same methodology can be used for optimizing other mass spectrometers. Ten picomoles of digest is needed for detection of glycopeptides in the present study, which uses 180-μm ID columns for on-line chromatographic fractionation of digests, but sub-picomole sensitivity is anticipated with the use of smaller columns and lower flow rates.
Joshi HJ, Harrison MJ, Schulz BL, Cooper CA, Packer NH, Karlsson NG. Development of a mass fingerprinting tool for automated interpretation of oligosaccharide fragmentation data. Proteomics 4;2004:1650–1664. [PubMed]
A computerized method for automated identification of separated oligosaccharides is described. The method uses masses of collision-induced fragment ions, and involves matching experimental MS/MS data with theoretical fragmentation of glycan structures in a suitable database. It returns a list of candidate oligosaccharide structures ranked by score. The scores are calculated using algorithms designed to estimate the degree of ambiguity of the sequence assigned from the fragments by database matching, and the degree to which the assigned sequence is supported by the relative intensities of the assigned fragments. Use of the software is intended to avoid some of the hard work of manual spectral interpretation. When tested using 130 MS/MS spectra from identified oligosaccharides, the routine ranks the correct structure top of the list in 78% of cases and second on the list in an additional 17% of cases. A web-based implementation of the software called GlycosidIQ is available from Proteome Systems (Sydney, Australia), where it can be used in conjunction with GlycoSuiteDB, a curated and annotated database which contains over 8000 published structures.
Doré K, Dubus S, Ho H-A, Lévesque I, Brunette M, Corbeil G, Boissinot M, Boivin G, Bergeron MG, Boudreau D, Leclerc M. Fluorescent polymeric transducer for the rapid, simple, and specific detection of nucleic acids at the zeptomole level. Journal of the American Chemical Society 126;2004:4240–4244. [PubMed]
Ultra-high sensitivity detection of DNA duplex strands is achieved without chemical labeling reactions. The method employs a fluorescent, water-soluble, cationic polymer, polythiophene, that interacts electrostatically with negatively charged nucleic acids. Duplexes formed between the polythiophene and single-stranded DNA aggregate and the polymer’s fluorescence is quenched. Formation of triplex strands with duplex DNA, however, produces increased fluorescence. Measurements can be completed simply and affordably, in under one hour. The detection limit estimated for a pair of perfectly matched 20-mers in a volume of 150 μL is 0.54 zmol (310 molecules). The method is quantitative, and mismatches can be readily distinguished. The system is shown to detect as few as 750 copies of an influenza A virus RNA genome in a procedure taking less than 30 minutes using a 32-mer oligonucleotide recognizing all type-A strains. The development of simple, cost-effective diagnostic tools not requiring amplification is envisaged.
Hong JW, Studer V, Hang G, Anderson WF, Quake SR. A nanoliter-scale nucleic acid processor with parallel architecture. Nature Biotechnology 22;2004:435–439.
Microfluidic chips for purification of mRNA and DNA from small numbers of mammalian or bacterial cells are described. Cell lysis and nucleic acid isolation and recovery are performed in an automated fashion without pre- or post-sample treatment. From single NIH 3T3 cells, detectable amounts of a high abundance mRNA (β-actin) and a moderate-abundance mRNA (zinc-finger OZF transcript) are recovered. Parallel chip architectures capable of processing several samples simultaneously are demonstrated. Among the applications anticipated are environmental and medical diagnostics, analysis of microcultures and of slow-growing or unculturable bacteria, rapid analysis of clone inserts and cell lines in genetic engineering studies, and the generation of subtractive libraries from pairs of single cells.
Zhang Y, Skolnick J. Automated structure prediction of weakly homologous proteins on a genomic scale. Proceedings of the National Academy of Sciences, USA. 101;2004:7594–7599.
An approach to protein tertiary structure prediction called “threading assembly refinement” is described. Threading is a process that predicts structure not just by aligning the target sequence to an evolutionarily related template protein with solved structure, but goes further by matching sequences to proteins adopting similar folds, where the target and template proteins need not be evolutionarily related. The new approach is applied to a benchmark set of 1489 medium-sized proteins in the Protein Data Bank. Excluding homologs, 927 templates identified by the threading algorithm, PROSPECTOR_3, have rms deviation from native of less than 6.5 Å with ≈80% alignment coverage. When template fragments are assembled together according to standard criteria, the number of proteins so aligned increases to 1172. Using confidence criteria established in this Protein Data Bank benchmark, the approach is applied to 1360 medium-sized ORFs in E. coli. Of these, the structures for 920 are predicted with high accuracy. The ability to fold two-thirds of all nonhomologous or weakly homologous proteins of less than 200 residues represents significant progress in solving the protein structure prediction problem.
Bonneau R. Baliga NS, Deutsch EW, Shannon P, Hood L. Comprehensive de novo structure prediction in a systems-biology context for the archaea Halobacterium sp. NRC-1. Genome Biology 4;2004:R52.
This work seeks to provide a prototype for the development of a biological data integration system that focuses on predicting function through de novo protein structure predictions. Rosetta de novo structure prediction is used to predict structures for 1185 proteins and protein domains of less than 150 residues in length in Halobacterium NRC-1. The predicted structures are then searched against the Protein Data Bank to identify similarities and extrapolate putative functions. These results are analyzed in the context of a model of functional protein associations that encompasses coordinated mRNA expression changes from microarray experiments among other data. The functions of several proteins are illuminated by this effort, and the number of unannotated proteins encoded by the genome of this bacterium is correspondingly reduced.
Smirnov IP, Zhu X, Taylor T, Huang Y, Ross P, Papayanopoulos IA, Martin SA, Pappin DJ. Suppression of α-cyano-4-hydroxycinnamic acid matrix clusters and reduction of chemical noise in MALDI-TOF mass spectrometry. Analytical Chemistry 76;2004:2958–2965. [PubMed]
It is a common practice to wash MALDI target spots made using α-cyano-4-hydroxycinnamic acid matrix with water to remove sodium and potassium and hence reduce the prominence of matrix cluster ions in the low mass range. Inclusion of ammonium salts in the washing solution is shown here to improve the efficiency of the wash treatment. Further reduction of chemical background and enhancement of peptide signal strength is achieved by incorporation of ammonium salts in the matrix, with monoammonium phosphate giving the best results. The combined effect is a 3–5-fold improvement in sensitivity.
Richert S, Luche S, Chevallet M, Van Dorsselaer A, Leize-Wagner E, Rabilloud T. About the mechanism of interference of silver staining with peptide mass spectrometry. Proteomics 4;2004:909–916. [PubMed]
The observation is reported that when proteins are detected after gel electrophoresis by silver staining using a protocol that employs formaldehyde in the developer, poorer peptide mass fingerprinting results are obtained if the stained gel is left in a water bath for 2 days before further processing than if processing is begun immediately. This suggests that the formaldehyde plays a role in interference with peptide mass fingerprint analysis. Use of carbohydrazide as the developing reagent avoids this problem, although it results in higher background staining and poorer staining homogeneity.
Cooper JW, Lee CS. Integrated and ultrasensitive gel protein identification. Analytical Chemistry 76;2004:2196–2202. [PubMed]
The benefits of combining a high-efficiency method for extraction of proteins from 2D gels and a high-efficiency method for digestion with trypsin are described. Proteins are stained with Sypro Red (Molecular Probes, Eugene, OR) for enhanced detection sensitivity. They are then electrokinetically extracted through a fused silica capillary placed in contact with the gel surface, using unbuffered water at the gel/capillary interface to concentrate the protein by a stacking mechanism. The protein is then passed through a miniature trypsin reactor consisting of a capillary fitting containing a PVDF membrane disk on which trypsin is immobilized. Digests from the reactor outflow are applied directly to a MALDI target. The extraction and digestion process is complete in less than 5 min. Standard proteins are identified with high sequence coverage at 1 ng protein loading on the gel, and identification of low abundance proteins from 2D gel separations of yeast proteins is demonstrated.
Schroeder MJ, Shabanowitz J, Schwartz JC, Hunt DF, Coon JJ. A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. Analytical Chemistry 76;2004:3590–3598. [PubMed]
Collision-induced dissociation (CID) of phosphopeptides often produces product ions dominated by the neutral loss of phosphoric acid. In ion-trap mass spectrometers, the resulting spectra may lack information from peptide backbone cleavages that are necessary to identify the peptide. In this paper, the instrument control software is altered to allow a series of sequential, user-defined ion activations, each targeting a m/z window of approximately 3 m/z units, that are used to activate product ions derived from the neutral loss of phosphoric acid for further CID. The fragments resulting from these activations are all stored together to provide a composite pseudo-MSn spectrum that contains enhanced amino acid sequence information. Targeted for activation are ions of 2+ charge state showing neutral loss of 49 [H3PO4], 58 [H3PO4 + H2O], and 98 [2 H3PO4]; and ions of 3+ charge state showing neutral loss of 32.6 [H3PO4], 38.6 [H3PO4+H2O], and 65 [2H3PO4]. The impact of this procedure on nonphosphorylated peptides is negligible. This method is shown to enhance the identification of phosphopeptides in many cases.
Syka JEP, Coon JJ, Schroeder MJ, Shabanowitz J, Hunt DF. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proceedings of the National Academy of Sciences, USA. 101;2004:9528–9533.
Electron capture dissociation (ECD) of peptides, a process that results from the interaction between peptide ions and thermal electrons to produce fragment ions predominantly of the c- and z-types, is superior to collision-induced dissociation (CID), which produces ions predominantly of the b- and y-types, in its preservation of post-translational modifications. Electron capture dissociation is used only in Fourier transform ion cyclotron resonance (FTICR) mass spectrometers because it cannot be implemented in devices that use RF fields to trap ions. This paper describes methodology for producing ECD-like spectra in a linear quadrupole ion-trap. The instrument is fitted with a second source that supplies negatively charged reagent ions that can transfer electrons to peptide ions during ion/ion interactions. c-Ions and z-ions are produced in a process called electron transfer dissociation. The utility of the system is demonstrated with anthracene reagent anions. Results for a variety of peptides that would be difficult to sequence by CID are shown. Methods are also suggested for using the instrumentation to characterize the primary structure of intact proteins.
Syka JEP, Marto JA, Bai DL, Horning S, Senko MW, Schwartz JC, Ueberheide B, Garcia B, Busby S, Muratore T, Shabanowitz J, Hunt DF. Novel linear quadrupole ion trap/FT mass spectrometer: Performance characterization and use in the comparative analysis of histone H3 post-translational modifications. Journal of Proteome Research 3;2004:621–626. [PubMed]
The construction and performance of a prototype tandem hybrid mass spectrometer consisting of a linear Paul-type quadrupole ion-trap interfaced with a Fourier transform mass spectrometer Penning trap is described. The hybrid instrument functions to provide rapid and automated MS and MS/MS data similar to the data-dependent scanning of conventional ion-trap mass spectrometers. A commercial version of the instrument has become available from Thermo Electron that operates with on-line HPLC, provides a resolution of 100,000 at 1 scan/sec and a mass accuracy of 1–2 ppm with external calibration. MS/MS spectra are acquired using either the linear ion-trap or the Penning trap.
Scherl A, Francois P, Converset V, Bento M, Burgess JA, Sanchez J-C, Hochstrasser DF, Schrenzel J, Corthals GL. Nonredundant mass spectrometry: A strategy to integrate mass spectrometry acquisition and analysis. Proteomics 4;2004:917–927.
MALDI-TOF/TOF mass spectrometers are now used routinely to identify proteins based upon MS and MS/MS spectra acquired from digests. In the present paper, initial acquisition of MS/MS data from each sample is limited to a small number of spectra prior to database searching. Acquisition of subsequent MS/MS spectra is then restricted in a data-dependent manner to precursors not expected from the sequence of the identified protein(s). This strategy saves time on data acquisition and validation, increases the number of proteins identified in mixtures, and increases sequence coverage by identifying unexpected peptides that may result from post-translational modifications.
Han X, Yang J, Cheng H, Ye H, Gross RW. Toward fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry. Analytical Biochemistry 330;2004:317–331. [PubMed]
Methodology is described for fingerprinting cellular lipids that includes most of the major cellular lipid classes and many of the minor ones, representing greater than 80% of the total lipid mass altogether. The methodology is applied to mouse hepatic tissue. Lipids are prepared by simple chloroform extraction. Chromatographic separation is not employed. Analyte-reagent ion interactions in the ion source are used to select different classes of lipid for study in turn: negative ion mass spectrometry of lithium-coordinated species at neutral pH selects for anionic lipids; negative ion mass spectrometry in the presence of LiOH is used to study ethanolamine glycerophopholipids; and positive ion mass spectrometry in the presence of LiOH selects for phosphatidylcholine and triacylglycerol species. Under each set of conditions, neutral loss or precursor ion scanning is used to focus on each class or subgroup of the ionized species, and the different isobaric species in each ion peak can be identified and quantitated.
Nordström A, Tarkowski P, Tarkowska D, Dolezal K, Åstot C, Sanderberg G, Moritz T. Derivatization for LC-electrospray ionization-MS: A tool for improving reversed-phase separation and ESI responses of bases, ribosides, and intact nucleotides. Analytical Chemistry 76;2004:2869–2877. [PubMed]
Very polar compounds are analyzed by reverse phase liquid chromatography-electrospray mass spectrometry in a method involving esterification of free hydroxyl groups on the analytes with propionyl or benzoyl acid anhydrides. The method is applied to the plant hormones, cytokinins, which are adenine bases substituted at the N-6 position with an isoprenoid side-chain. Cytokinins occur as free bases, nucleosides, nucleotides, and glucosides. Esterification has the effects of increasing the retention on reverse-phase columns (ion-pairing reagents are not needed owing to the increased analyte hydrophobicity), and improving electrospray response owing to improved surface activity. Detection limits for propionylated cytokinins were in the upper attomole to lower femtomole range, an improvement of 10–100 compared with previously reported figures.
Sartor M, Schwanekamp J, Halbleib D, Mohamed I, Karyala S, Medvedovic M, Tomlinson CR. Microarray results improve significantly as hybridization approaches equilibrium. BioTechniques 36;2004:790–796. [PubMed]
Using dual-channel, long oligonucleotide microarrays, increasing hybridization times from 18 h to 42 h and to 66 h is found to increase significantly the numbers of spots detecting target, signal-to-noise ratios, numbers of differentially expressed genes, and correlations among replicate arrays. The improvement is especially marked when lower-than-optimal quantities of target are used.
Etienne W, Meyer MH, Peppers J, Meyer RA Jr. Comparison of mRNA gene expression by RT-PCR and DNA microarray. BioTechniques 36;2004:618–626. [PubMed]
This study compares the quantitation of mRNA species obtained using Affymetrix DNA microarrays with that measured using reverse transcription PCR (RT-PCR). Twenty-six genes are subject to comparison in the context of work on the healing of bone fracture in the rat. Ten samples are studied for each gene, representing the progress of the healing process, giving a wide range of expression levels that vary though the time-course represented by the sample series. The results from the two techniques are correlated. Correlation coefficients (r) ranging from −0.48 to +0.93 are measured, with a mean of 0.28. Good agreement between the methods is observed for genes with moderate expression levels. However, genes with very high or low expression levels, and genes for which there is a large separation between the location of the PCR and the microarray probes, give poorer agreement.
Ramachandran N, Hainsworth E, Bhullar B, Eisenstein S, Rosen B, Lau AY, Walter JC, LaBaer J. Self-assembling protein microarrays. Science 305;2004:86–90. [PubMed]
The manufacture of protein arrays for studying functional interactions with other molecules such as drugs, antibodies, nucleic acids, lipids, or other proteins is rendered difficult by the challenges of high-throughput protein production and instability of proteins before or after spotting. These issues are addressed in the present paper by printing cDNAs onto glass microscope slides and then translating them in situ using a suitable cell-free translation system to form target proteins. The proteins are immobilized on the array via C-terminal GST tags. This approach avoids the need to express and purify proteins separately, and produces proteins at the time of assay, eliminating concerns about protein stability during storage. Protein arrays manufactured in this way are used to map pairwise interactions between 29 proteins involved in DNA replication initiation. The spots contain 4–29 fmol, a sevenfold spread that falls within the range observed with spotted protein arrays. The results confirm and extend existing information about the regulation of the system, and, using deletion fragments of the initiation factor Cdt1, map sites of interaction between this protein and its binding partner, geminin.
Hurst GB, Lankford TK, Kennel SJ. Mass spectrometric detection of affinity purified crosslinked peptides. Journal of the American Society for Mass Spectrometry 15;2004:832–839. [PubMed]
A commercially available biotinylated cross-linking reagent is used to provide affinity-based enrichment of crosslinked peptides from digests of proteins whose interaction is being studied. The crosslinker is sulfo-succinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate (sulfo-SBED) from Pierce, (Rockland, IL). This is a heterobifunctional reagent with an N-hydroxysuccinimide (NHS) ester that reacts with amines, and a photoactivatable aryl azide that typically inserts into N-H or C-H bonds with the loss of N2. The reagent is tested by intramolecular crosslinking of neurotensin. The predicted crosslinked product is identified, along with several other products. Specificity of the affinity purification is optimized by incorporating an additional blocking step and washes, and recovery is optimized by minimization of the amount of avidin beads employed.
Denison C, Kodadek T. Toward a general chemical method for rapidly mapping multi-protein complexes. Journal of Proteome Research 3;2004:417–425. [PubMed]
The performance of a novel cross-linking reagent, tris(2,2′-bipyridyl)ruthenium (II) dication (Ru(II) (bpy)32+), is assessed by mapping the subunits of the 20 S proteasome, a multisubunit complex whose architecture is known from X-ray crystallography. When photolyzed for only 0.5 sec in the presence of ammonium persulfate, Ru(II)(bpy)32+ is photo-oxidized to (Ru(II)(bpy)33+. This intermediate oxidizes tyrosine and tryptophan residues on a protein, producing radical species that couple rapidly to nearby functional groups. The ruthenium complex thereby acts as a catalyst for the formation of zero-length crosslinks between neighboring functional groups. Subunit–subunit interactions in the proteasome are documented by recovery of crosslinked subunits from SDS-polyacrylamide gels and then identifying them by mass spectrometry. All crosslinks documented are between subunits known to be adjacent in the complex. All but 2 of the 14 subunits are represented among the crosslinked products. However, not all the expected contacts are detected, indicating that the use of other crosslinkers may provide complementary information.
Rasa M, Philipse AP. Evidence for a macroscopic electric field in the sedimentation profiles of charged colloids. Nature 429;2004:857–860. [PubMed]
Using charged silica particles in ethanol, “inflation” of equilibrium sedimentation/diffusion profiles is observed during ultracentrifugation, with greater particle density (concentration) at the top of the liquid column and lesser density at the bottom, as though the particles weigh less than they actually do. This effect is ascribed to a Donnan-type electrical potential gradient due to the gradient in the density of the charged particles along the length of the liquid column. This effect is a possible source error in molecular weight measurements of protein molecules undertaken by analytical ultracentrifugation in solutions of low ionic strength. See also commentary by Warren P. Electrifying effects in colloids. Nature 429;2004:822. [PubMed]
Myszka DG. Analysis of small-molecule interactions using Biacore S51 technology. Analytical Biochemistry 329;2004:316–323. [PubMed]
The Biacore S51 is a new surface plasmon resonance instrument from Biacore AB (Uppsala, Sweden) that is designed to support analysis of interactions between proteins and small molecules by incorporating increased sensitivity, the ability to handle large numbers of samples, and automated data processing. It also incorporates a new flow cell design that provides two reaction surfaces and a reference surface, all within the same liquid compartment, to improve data quality and permit the analysis of faster binding interactions. This instrument is tested using a set of small molecule inhibitors of the enzyme carbonic anhydrase II varying in size from 340 to 95 Da and displaying an approximately 10,000-fold range of affinities for the enzyme. Binding responses are shown to be highly reproducible and changes in response as low as 0.50 RU (0.5 pg/mm2) are reliably determined.
Cannon MJ et al. Comparative analysis of a small molecule/enzyme interaction by multiple users of Biacore technology. Analytical Biochemistry 330;2004:98–113. [PubMed]
A benchmark study to educate users of Biacore instruments in proper instrument maintenance and experimental design for small molecules is described. Thirty-six laboratories provide data for the interaction between a small molecule inhibitor, acetazolamide, and the enzyme carbonic anhydrase II. Participants adhere to prescribed protocols in which the enzyme is immobilized at three different densities and the inhibitor is supplied in a 250-fold range of concentrations. The interaction is a difficult one to analyze because the ligand is a small molecule demanding high sensitivity to detect, and the interaction is rapid and mass transport limited. Although most participants collect data of adequate quality, two areas of significant deviation in the magnitude of binding constants are identified. The first is due to variation in the amount of enzyme immobilized. This is probably the consequence to each participant providing his/her own immobilization reagents. The second is due to variation in the quality of the response data (spikes and irreproducible binding values). This is probably due to instrument contamination or malfunctioning pumps.
Karp NA, Krell DP, Lilley KS. Determining a significant change in protein expression with DeCyderTM during a pair-wise comparison using two-dimensional difference gel electrophoresis. Proteomics 4;2004:1421–1432. [PubMed]
In two-dimensional difference gel electrophoresis (DIGE), protein expression analysis is performed by multiplexing samples labeled with different cyanine dyes and examining spot patterns for expression changes using Amersham’s DeCyder software. The recognition of significant changes in the level of protein expression depends in part on assessing the level of “same sample” variance. In the present study, the distribution of volume ratios in same sample comparisons is shown to exhibit a high degree of asymmetry, heavy bias at low volume values due to differences in dye background, and significant gel-to-gel variation in spot volume ratio distributions. In the face of these problems, the normalized data calculated by DeCyder are unable to support the calculation of thresholds for identification of expression changes with 90 or 95% confidence limits that hold from gel to gel. An alternative normalization routine is presented that, when used in combination with a standardizing function, improves the success of DIGE data analysis by supporting identification of expression changes with 90% confidence in a gel-independent fashion.
Pieper R, Gatlin CL, McGrath AM, Makusky AJ, Mondal M, Seonarain M, Field E, Schatz CR, Estock MA, Ahmed N, Anderson NG, Steiner S. Characterization of the human urinary proteome: A method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots. Proteomics 4;2004:1159–1174. [PubMed]
Methods for preparing urinary proteins for proteomic comparisons using 2D electrophoresis are described. Urine, to which protease inhibitors have been added, is concentrated, and fractions corresponding to proteins less than and greater than 30 kDa are collected by gel filtration chromatography. Albumin and immunoglobulin are removed from the fraction containing larger proteins. The resulting pools are subjected to 2D electrophoresis. Using mass spectrometry, 420 spots are successfully identified and shown to be derived from 150 different genes.
Abdolzade-Bavil A, Hayes S, Goretzki L, Kröger M, Anders J, Hendriks R. Convenient and versatile subcellular extraction procedure, that facilitates classical protein expression profiling and functional protein analysis. Proteomics 4;2004:1397–1405. [PubMed]
Methodology for separating cells lysates into fractions corresponding to cytosolic, membrane/organelle, soluble/DNA-associated nuclear, and cytoskeletal pools is described. The quality of the procedure is evaluated by morphology, and by marker analysis. The procedure is commercially available as ProteoExtract Subcellular Proteome Extraction from Calbiochem (San Diego, CA).
Resing KA, Meyer-Arendt K, Mendoza AM, Aveline-Wolf LD, Jonscher KR, Pierce KG, Old WM, Cheung HT, Russell S, Wattawa JL, Goehle GR, Knight RD, Ahn NG. Improving reproducibility and sensitivity in identifying human proteins by shotgun proteomics. Analytical Chemistry 76;2004:3556–3568. [PubMed]
When validation of peptide assignments is sought from threshold criteria for statistical scores generated by the standard search programs, Sequest and Mascot, only some 50%–60% of MS/MS spectra from multiply charged peptide ions are shown to be capable of yielding identities. Increased sensitivity and accuracy are shown to result from integrating the results obtained with the two database search programs. Improvement in distinguishing correct from incorrect assignments is also achieved by using scores describing the quality of the fragmentation spectrum, and by including chemical criteria deduced, for example, from chromatographic behavior. These approaches improve performance to the level of 4.2% false positives and 8% false negatives. Increased database size—due, for example, to selecting increased mass tolerance, which allows for nontryptic or missed cleavages as well as variable modifications—is shown to result in increased thresholds for validating Sequest and Mascot scores, indicating the need for focused searches.