DirecTag: accurate sequence tags from peptide MS/MS through statistical scoring

In shotgun proteomics, tandem mass spectra of peptides are typically identified through database search algorithms such as Sequest. We have developed DirecTag, an open-source algorithm to infer partial sequence tags directly from observed fragment ions. This algorithm is unique in its implementation of three separate scoring systems to evaluate each tag on the basis of peak intensity, m/ z fidelity, and complementarity. In data sets from several types of mass spectrometers, DirecTag reproducibly exceeded the accuracy and speed of InsPecT and GutenTag, two previously published algorithms for this purpose. The source code and binaries for DirecTag are available from http://fenchurch.mc.vanderbilt.edu.     

A rapid and accurate UPLC/MS/MS method for the determination of benzodiazepines in human urine

A rapid, sensitive, and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) assay method for simultaneous determination of 13 benzodiazepine compounds in human urine was developed and validated. Aliquots of 0.5 mL of urine specimens were used for the analysis and the benzodiazepines were extracted by single step methanol (containing 0.2% formic acid) precipitation and then separated on a BEH C18 (50 mm × 2.1 mm, 1.7 μm) analytical column with the temperature maintained at 45°C. The mobile phases consisted of methanol and water (both containing 0.2% formic acid) and the flow rate was 0.4 mL/min. The TQ detector, equipped with an electrospray ionization ion source, was set up with a positive mode. The acquisitions were performed in multiple-reaction monitoring (MRM) and the limit of quantification was 20 ng/mL for all of the 13 compounds. The low limits of detections (LODs) of the benzodiazepines in this method were between 0.5 and 2 ng/mL. The chromatographic separation time was 4 min and calibration curves in human urine were generated over the range of 20-2000 ng/mL. The method validation parameters such as accuracy, precision, carryover, recovery, stability, and specificity for all of the 13 compounds were within the acceptable range. This method is suitable for the high throughput screening of benzodiazepines in clinical laboratories.     

Mitochondrial phosphoproteome revealed by an improved IMAC method and MS/MS/MS

IMAC in combination with mass spectrometry is a promising approach for global analysis of protein phosphorylation. Nevertheless this approach suffers from two shortcomings: inadequate efficiency of IMAC and poor fragmentation of phosphopeptides in the mass spectrometer. Here we report optimization of the IMAC procedure using (32)P-labeled tryptic peptides and development of MS/MS/MS (MS3) for identifying phosphopeptide sequences and phosphorylation sites. The improved IMAC method allowed recovery of phosphorylated tryptic peptides up to approximately 77% with only minor retention of unphosphorylated peptides. MS3 led to efficient fragmentation of the peptide backbone in phosphopeptides for sequence assignment. Proteomics of mitochondrial phosphoproteins using the resulting IMAC protocol and MS3 revealed 84 phosphorylation sites in 62 proteins, most of which have not been reported before. These results revealed diverse phosphorylation pathways involved in the regulation of mitochondrial functions. Integration of the optimized batchwise IMAC protocol with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation.     

An evaporation-free solid-phase extraction method for rapid and accurate analysis of sumatriptan in human plasma by LC-MS/MS

An evaporation-free solid-phase extraction (SPE) method was developed and validated for sumatriptan. High organic washing (50% methanol) and low organic elution (20% methanol) were used and the recovery was greater than 92%. The eluate was injected into a C18 column without evaporation and reconstitution. Sumatriptan was monitored in positive ion mode with mass transition of m/z 296.4-58.1 amu. The calibration curve was 1-100 ng/mL (r>or=0.9923). The inter-day and intra-day precisions ranged from 4.53 to 9.12% and 1.72 to 6.93%, respectively. This method features reduced cost and pollution, clean extract, high speed, and most importantly overall method reliability.     

RAId_aPS: MS/MS analysis with multiple scoring functions and spectrum-specific statistics

Statistically meaningful comparison/combination of peptide identification results from various search methods is impeded by the lack of a universal statistical standard. Providing an E-value calibration protocol, we demonstrated earlier the feasibility of translating either the score or heuristic E-value reported by any method into the textbook-defined E-value, which may serve as the universal statistical standard. This protocol, although robust, may lose spectrum-specific statistics and might require a new calibration when changes in experimental setup occur. To mitigate these issues, we developed a new MS/MS search tool, RAId_aPS, that is able to provide spectrum-specific-values for additive scoring functions. Given a selection of scoring functions out of RAId score, K-score, Hyperscore and XCorr, RAId_aPS generates the corresponding score histograms of all possible peptides using dynamic programming. Using these score histograms to assign E-values enables a calibration-free protocol for accurate significance assignment for each scoring function. RAId_aPS features four different modes: (i) compute the total number of possible peptides for a given molecular mass range, (ii) generate the score histogram given a MS/MS spectrum and a scoring function, (iii) reassign E-values for a list of candidate peptides given a MS/MS spectrum and the scoring functions chosen, and (iv) perform database searches using selected scoring functions. In modes (iii) and (iv), RAId_aPS is also capable of combining results from different scoring functions using spectrum-specific statistics. The web link is http://www.ncbi.nlm.nih.gov/CBBresearch/Yu/raid_aps/index.html. Relevant binaries for Linux, Windows, and Mac OS X are available from the same page.     

MALDI‐TOF and nESI Orbitrap MS/MS identify orthogonal parts of the phosphoproteome

Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows. In this study, we compared tandem mass spectrometry (MS/MS) on matrix assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) and nano‐electrospray ionization (nESI) Orbitrap instruments with respect to their ability to identify phosphopeptides from complex proteome digests. Phosphopeptides were enriched from tryptic digests of cell lines using Fe‐IMAC column chromatography and subjected to LC‐MS/MS analysis. We found that the two analytical workflows exhibited considerable orthogonality. For instance, MALDI‐TOF MS/MS favored the identification of phosphopeptides encompassing clear motif signatures for acidic residue directed kinases. The extent of orthogonality of the two LC‐MS/MS systems was comparable to that of using alternative proteases such as Asp‐N, Arg‐C, chymotrypsin, Glu‐C and Lys‐C on just one LC‐MS/MS instrument. Notably, MALDI‐TOF MS/MS identified an unexpectedly high number and percentage of phosphotyrosine sites (～20% of all sites), possibly as a direct consequence of more efficient ionization. The data clearly show that LC‐MALDI MS/MS can be a useful complement to LC‐nESI MS/MS for phosphoproteome mapping and particularly so for acidic and phosphotyrosine containing peptides.     

Highly selective and rapid enrichment of phosphorylated peptides using gallium oxide-coated magnetic microspheres for MALDI-TOF-MS and nano-LC-ESI-MS/MS/MS analysis

In this work, we present, to our knowledge, the first demonstration of the utility of iron oxide magnetic microspheres coated with gallium oxide for the highly selective enrichment of phosphopeptide prior to mass spectrometric analysis. These microspheres that we prepared not only have a shell of gallium oxide, giving them a high-trapping capacity for the phosphopeptides, but also their magnetic property enables easy isolation by positioning an external magnetic field. Tryptic digest products of phosphoproteins including beta-casein, ovalbumin, casein, as well as five protein mixtures were used as the samples to exemplify the feasibility of this approach. In very short time (only 0.5 min), phosphopeptides sufficient for characterization by MALDI-TOF-MS were selectively enriched by the Ga(2)O(3)-coated Fe(3)O(4) microspheres. The performance of the Ga(2)O(3)-coated Fe(3)O(4) microspheres were further compared with Fe(3+)-immobilized magnetic silica microspheres, commercial Fe(3+)-IMAC resin, and TiO2 beads for enrichment of peptides originating from tryptic digestion of beta-casein and BSA with a molar ratio of 1:50, and the results proved a stronger selective ability of Ga(2)O(3)-coated Fe(3)O(4) microspheres over the other materials. Finally, the Ga(2)O(3)-coated Fe(3)O(4) microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. All results show that Ga(2)O(3)-coated Fe(3)O(4) microsphere is an effective material for selective isolation and concentration of phosphopeptides.     

A simple and accurate GC/MS method for the quantitative analysis of diacetyl in beer and wine

A GC/MS method for the quantification of diacetyl is described. Diacetyl is derivatized with 4,5-dichloro-1,2-diaminobenzene to form 6,7-dichloro-2,3-dimethylquinoxaline (DCDMQ). The derivative is extracted in benzene and quantified by GC/MS. Formation of DCDMQ is linearly correlated with diacetyl concentration. The method is rapid, sensitive (determination limit 0.0005 g/mL) precise (standard error < 2%), and accurate (recovery of diacetyl 91.5% + 1.5%).     

New tools for quantitative phosphoproteome analysis.

Recent advances in analytical methods, particularly in the area of mass spectrometry, have brought the field of proteomics to the forefront in biological science. The ultimate goal of proteomics--to characterize proteins expressed within a cell under a specific set of conditions--is daunting due to the complexity and dynamic nature the of protein population within the cell. While much of the effort has focused on developing methods to identify expressed proteins, the identification of posttranslational modifications is equally important for comprehensive proteome characterization. Of all the known posttranslational modifications, phosphorylation arguably plays the largest role in the context of cellular homeostasis. This review discusses some of the recent progress made in the development of techniques not only to identify, but also to quantitatively determine sites of phosphorylation.     

An evaluation, comparison, and accurate benchmarking of several publicly available MS/MS search algorithms: sensitivity and specificity analysis

MS/MS and associated database search algorithms are essential proteomic tools for identifying peptides. Due to their widespread use, it is now time to perform a systematic analysis of the various algorithms currently in use. Using blood specimens used in the HUPO Plasma Proteome Project, we have evaluated five search algorithms with respect to their sensitivity and specificity, and have also accurately benchmarked them based on specified false-positive (FP) rates. Spectrum Mill and SEQUEST performed well in terms of sensitivity, but were inferior to MASCOT, X!Tandem, and Sonar in terms of specificity. Overall, MASCOT, a probabilistic search algorithm, correctly identified most peptides based on a specified FP rate. The rescoring algorithm, PeptideProphet, enhanced the overall performance of the SEQUEST algorithm, as well as provided predictable FP error rates. Ideally, score thresholds should be calculated for each peptide spectrum or minimally, derived from a reversed-sequence search as demonstrated in this study based on a validated data set. The availability of open-source search algorithms, such as X!Tandem, makes it feasible to further improve the validation process (manual or automatic) on the basis of "consensus scoring", i.e., the use of multiple (at least two) search algorithms to reduce the number of FPs. complement.     

A strategy for the rapid identification of phosphorylation sites in the phosphoproteome

Edman phosphate ((32)P) release sequencing provides a high sensitivity means of identifying phosphorylation sites in proteins that complements mass spectrometry techniques. We have developed a bioinformatic assessment tool, the cleavage of radiolabeled protein (CRP) program, which enables experimental identification of phosphorylation sites via (32)P labeling and Edman degradation of cleaved proteins obtained at femtomole levels. By observing the Edman cycle(s) in which radioactivity is found, candidate phosphorylation sites are identified by determining which residues occur at the observed number of cycles downstream from a peptide cleavage site. In cases where more than one residue could be responsible for the observed radioactivity, additional experiments with cleavage reagents having alternative specificities may resolve the ambiguity. Given a protein sequence and a cleavage site, CRP performs these experiments in silico, identifying resolved sites based on user-supplied experimental data, as well as suggesting combinations of reagents for additional analyses. Analysis of the PhosphoBase protein sequence database suggests that CRP data from two cleavage experiments can be used to identify unambiguously 60% of known phosphorylation sites. Data from additional cleavage experiments may increase the overall coverage to 70% of known sites. By comparing theoretical data obtained from the CRP program with (32)P release data obtained from an Edman sequencer, a known phosphorylation site was identified unambiguously and correctly. In addition, our results show that in vivo phosphorylation sites can be determined routinely by differential proteolysis analysis and Edman cycling with less than 1 fmol of protein and 1000 cpm.     

A multidimensional chromatography technology for in-depth phosphoproteome analysis

Protein phosphorylation is a post-translational modification widely used to regulate cellular responses. Recent studies showed that global phosphorylation analysis could be used to study signaling pathways and to identify targets of protein kinases in cells. A key objective of global phosphorylation analysis is to obtain an in-depth mapping of low abundance protein phosphorylation in cells; this necessitates the use of suitable separation techniques because of the complexity of the phosphoproteome. Here we developed a multidimensional chromatography technology, combining IMAC, hydrophilic interaction chromatography, and reverse phase LC, for phosphopeptide purification and fractionation. Its application to the yeast Saccharomyces cerevisiae after DNA damage led to the identification of 8764 unique phosphopeptides from 2278 phosphoproteins using tandem MS. Analysis of two low abundance proteins, Rad9 and Mrc1, revealed that approximately 50% of their phosphorylation was identified via this global phosphorylation analysis. Thus, this technology is suited for in-depth phosphoproteome studies.     

Web-based MS/MS data analysis

This tutorial focuses on three MS/MS data analysis programs currently available via a web interface: Mascot, Phenyx and X!Tandem. Although these programs process the same input and often produce comparable outputs, subtle differences remain. The use of parameters that are requested in the on-line forms and the subsequent interpretation of results are illustrated and explained via a single example.     

An accurate quantitative LC/ESI-MS/MS method for sirolimus in human whole blood

Sirolimus is a widely used immunosuppressant that requires therapeutic drug monitoring (TDM). We optimized a preanalytical procedure that allows for the accurate quantiation of sirolimus in whole blood by LC/ESI-MS/MS with minimal matrix effects. Sirolimus is highly lipophilic, and solvents containing greater than 50% methanol were required to maintain sirolimus recovery. The final pretreatment procedure developed consists of a zinc sulfate protein precipitation, an extraction using octadecyl silyl-silica gel for eliminating water-soluble and hydrophilic compounds, and HybridSPE cartridge treatment to eliminate phospholipids. Using this procedure prior to LC/ESI-MS/MS led to the accurate and reproducible quantitation of sirolimus in human whole blood. The linear range of detection was 0.5-50 ng/mL, a range appropriate for TDM, and the method demonstrated good repeatability and intermediate precision within this quantitative range. In order to investigate the quantitative performance of this method, we compared it to two commercially available sirolimus immunoassays and our previously reported LC/ESI-MS/MS method. The immunoassays gave consistently greater values for the sirolimus concentration, and this may be related to antibody cross-reactivity with sirolimus metabolites and/or other matrix effects. Although our procedure is too long to support real-time TDM for outpatients, it can serve as reference method to assess the performance of other analytical methods that are currently available or may be developed in the future.     

Liquid chromatography MALDI MS/MS for membrane proteome analysis

Membrane proteins play critical roles in many biological functions and are often the molecular targets for drug discovery. However, their analysis presents a special challenge largely due to their highly hydrophobic nature. We present a surfactant-aided shotgun proteomics approach for membrane proteome analysis. In this approach, membrane proteins were solubilized and digested in the presence of SDS followed by newly developed auto-offline liquid chromatography/matrix-assisted laser desorption ionization (LC/MALDI) tandem MS analysis. Because of high tolerance of MALDI to SDS, one-dimensional (1D) LC separation can be combined with MALDI for direct analysis of protein digests containing SDS, without the need for extensive sample cleanup. In addition, the heated droplet interface used in LC/MALDI can work with high flow LC separations, allowing a relatively large amount of protein digest to be used for 1D LC/MALDI which facilitates the detection of low abundance proteins. The proteome identification results obtained by LC/MALDI are compared to the gel electrophoresis/MS method as well as the shotgun proteomics method using 2D LC/electrospray ionization MS. It is demonstrated that, while LC/MALDI provides more extensive proteome coverage compared to the other two methods, these three methods are complementary to each other and a combination of these methods should provide a more comprehensive membrane proteome analysis.     

Compositional protein analysis of high density lipoproteins in hypercholesterolemia by shotgun LC-MS/MS and probabilistic peptide scoring

A protein of a biological sample is usually quantified by immunological techniques based on antibodies. Mass spectrometry offers alternative approaches that are not dependent on antibody affinity and avidity, protein isoforms, quaternary structures, or steric hindrance of antibody-antigen recognition in case of multiprotein complexes. One approach is the use of stable isotope-labeled internal standards; another is the direct exploitation of mass spectrometric signals recorded by LC-MS/MS analysis of protein digests. Here we assessed the peptide match score summation index based on probabilistic peptide scores calculated by the PHENYX protein identification engine for absolute protein quantification in accordance with the protein abundance index as proposed by Mann and co-workers (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231-1245). Using synthetic protein mixtures, we demonstrated that this approach works well, although proteins can have different response factors. Applied to high density lipoproteins (HDLs), this new approach compared favorably to alternative protein quantitation methods like UV detection of protein peaks separated by capillary electrophoresis or quantitation of protein spots on SDS-PAGE. We compared the protein composition of a well defined HDL density class isolated from plasma of seven hypercholesterolemia subjects having low or high HDL cholesterol with HDL from nine normolipidemia subjects. The quantitative protein patterns distinguished individuals according to the corresponding concentration and distribution of cholesterol from serum lipid measurements of the same samples and revealed that hypercholesterolemia in unrelated individuals is the result of different deficiencies. The presented approach is complementary to HDL lipid analysis; does not rely on complicated sample treatment, e.g. chemical reactions, or antibodies; and can be used for projective clinical studies of larger patient groups.     

一种快速精确编辑疱疹病毒基因组的方法

为了快速且准确地对疱疹病毒基因组进行基因敲除、插入或者点突变等修饰,通过同源重组将马立克氏病病毒 (MDV) 超强毒株Md5基因组克隆到细菌人工染色体 (BAC)。将筛选的阳性重组体DNA电转进DH10B菌株,用PCR及限制性片段多态分析 (RFLP) 方法鉴定含Md5全基因组的BAC克隆。将阳性重组体DNA转染入鸡胚成纤维细胞 (CEF),拯救出重组病毒,命名为Md5BAC。进一步利用Red酶介导的两步法基因重组技术构建MDVlorf10基因敲除毒株。为了验证被敲除基因功能的特异性,将lorf10插入原位点以构建基因复原毒株。将构建的重组毒株分别感染CEF细胞,用间接免疫荧光试验确认重组病毒均包装成功;病毒生长曲线结果表明,lorf10敲除不影响病毒的体外增殖。总之,这为其他疱疹病毒的基因组编辑提供了技术参考。     

ISHAPE: new rapid and accurate software for haplotyping

Background  

We have developed a new haplotyping program based on the combination of an iterative multiallelic EM algorithm (IEM), bootstrap resampling and a pseudo Gibbs sampler. The use of the IEM-bootstrap procedure considerably reduces the space of possible haplotype configurations to be explored, greatly reducing computation time, while the adaptation of the Gibbs sampler with a recombination model on this restricted space maintains high accuracy. On large SNP datasets (>30 SNPs), we used a segmented approach based on a specific partition-ligation strategy. We compared this software, Ishape (Iterative Segmented HAPlotyping by Em), with reference programs such as Phase, Fastphase, and PL-EM. Analogously with Phase, there are 2 versions of Ishape: Ishape1 which uses a simple coalescence model for the pseudo Gibbs sampler step, and Ishape2 which uses a recombination model instead.