Targeted tandem mass spectrometry for high-throughput comparative proteomics employing NanoLC-FTICR MS with external ion dissociation

Targeted tandem mass spectrometry (MS/MS) is an attractive proteomic approach that allows selective identification of peptides exhibiting abundance differences, e.g., between culture conditions and/or diseased states. Herein, we report on a targeted LC-MS/MS capability realized with a hybrid quadrupole-7 tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer that provides data-dependent ion selection, accumulation, and dissociation external to the ICR trap, and a control software that directs intelligent MS/MS target selection based on LC elution time and m/z ratio. We show that the continuous on-the-fly alignment of the LC elution time during the targeted LC-MS/MS experiment, combined with the high mass resolution of FTICR MS, is crucial for accurate selection of targets, whereas high mass measurement accuracy MS/MS data facilitate unambiguous peptide identifications. Identification of a subset of differentially abundant proteins from Shewanella oneidensis grown under suboxic versus aerobic conditions demonstrates the feasibility of such approach.     

Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry

Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. In recent years mass spectrometry has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The present study used immobilized metal affinity chromatography (IMAC) coupled with a linear ion trap mass spectrometer to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although this number of identified phosphoproteins is not large, the approach is still of interest because a series of conservative criteria were adopted in data analysis. We note that, although the binding of non-phosphorylated peptides to the IMAC column was apparent, the improvements in high-speed scanning and quality of MS/MS spectra provided by the linear ion trap contributed to the phosphoprotein identification. Further analysis demonstrated that MS/MS/MS analysis was necessary to exclude the false-positive matches resulting from the MS/MS experiments, especially for multiphosphorylated peptides. The use of the linear ion trap considerably enabled exploitation of nanoflow-HPLC/MS/MS, and in addition MS/MS/MS has great potential in phosphoproteome research of relatively complex samples.     

Tandem mass spectrometric accurate mass performance of time-of-flight and Fourier transform ion cyclotron resonance mass spectrometry: a case study with pyridine derivatives

The interpretation of mass spectra is a key process during compound identification, and the combination of tandem mass spectrometry (MS/MS) with high-accuracy mass measurements may deliver crucial information on the identity of a compound. Obtaining accurate mass data of fragment ions in MS/MS reveals the particular problem of mass calibration when a lockmass, which is frequently used to obtain accurate masses in MS, is absent. An alternative technique is to recalibrate the MS/MS spectrum using a reference MS/MS spectrum acquired under the same conditions. We have tested and validated this approach using a hybrid quadrupole/orthogonal acceleration reflectron-type time-of-flight (TOF) mass spectrometer. The results were compared with those obtained under similar conditions on a Fourier transform ion cyclotron resonance (FT-ICR) instrument. We found that the mass accuracy observed with such an "external" recalibration on the TOF instrument in MS/MS is identical to what can be obtained on a similar instrument operating in one-dimensional MS mode using the lockmass technique. However, mass accuracy in both cases is one order of magnitude inferior to that obtained using FTMS, and also inferior to that observed using sector field MS when operated at comparable resolution. Nevertheless, for small (<200 Da) molecules, this mass accuracy was still sufficient to have the "true" elemental composition identified as the first hit in about 70% of all cases. It was possible to elucidate the fragmentation mechanism of eight azaheterocycles containing a pyridine moiety, where the accurate mass data from the TOF instrument allowed distinction between two alternative fragmentation pathways.     

Protein identification from two-dimensional gel electrophoresis by connecting ZipTip with a home-assembled nanoflow system

Mass spectrometry (MS) is a powerful technique for protein identification in proteomic research. Two-dimensional gel electrophoresis (2-DE) combined with MS is a significant method for protein separation and identification. For protein identification, peptide sequencing is usually carried out by an effective but expensive nano-flow liquid chromatographic system combined to tandem mass spectrometry (MS/MS). However, protein identification based on such method is time-consuming, and contamination may occur as a result of column overloading. In this study, we establish an alternative nanoscale system for protein identification using MS/MS. The system consists of several devices that can be purchased from commercial sources and can be connected to an electrospray ionization quadrupole-time of flight (ESI-Q-TOF) MS in order to analyze proteins from 2D gels. This inexpensive strategy provides an attractive alternative method for rapid identification of proteins using a nanospray source. In addition, the device is disposable so that contamination is avoided. It is shown that peptide sequencing based on this device using ESI-Q-TOF MS is accomplished within 10 min.     

Structural analysis of drug-DNA adducts by tandem mass spectrometry

The utility of electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) for the characterisation of ligand-oligonucleotide adducts is demonstrated with adducts formed between the oligonucleotide 5'-CACGTG-3' and both a platinating agent, cis-diamminedichloroplatinum(II) (cisplatin), and an alkylating ligand, n-bromohexylphenanthridinium bromide (phenC6Br). We have demonstrated previously that negative ion MS/MS spectra of alkylated oligonucleotides show a highly specific fragmentation pathway that enables the site of binding of the ligand to be readily identified. In comparison, the positive ion ESI-MS/MS spectra reported here also show a single major fragmentation pathway, but the dominant ion is the protonated ligand-base adduct. MS/MS of this ion confirms the site on binding of the ligand to the guanine base. MS/MS spectra of cisplatin adducts show much less specific fragmentation than alkylated adducts, particularly in the negative ion mode. This suggests that the ESI-MS/MS spectra of ligand-DNA adducts are strongly influenced by the extent to which the ligand weakens the glycosidic bond in the residue to which it is bound. For platinating agents, which do not labilise the glycosidic bond, additional experiments involving MS/MS of source-generated product ions were required to enable isomeric adducts to be distinguished.     

Analysis of proteins from a glioma cell line by using micro-scale solution isoelectric focusing in combination with liquid chromatography/tandem mass spectrometry

In this study we have investigated whether micro-solution isoelectric focusing (microsol-IEF) can be used as a pre-fractionation step prior to liquid chromatography/tandem mass spectrometry (LC/MS/MS) and if extensive sample purification of the different fractions is required. We found that, in spite of the high concentrations of buffer and detergents, no clean up of the digested microsol-IEF fractions was necessary before analysis by LC/MS/MS. We also concluded that it is possible to identify at least twice as many proteins in a glioma cell lysate with the combination of microsol-IEF and LC/MS/MS than with LC/MS/MS alone. Furthermore, most of the proteins that were identified from one microsol-IEF fraction by using analytical narrow-range two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were also identified by LC/MS/MS. Finally, we used the combination of microsol-IEF and LC/MS/MS to compare two sample preparation methods for glioma cells and found that several nuclear, mitochondria, and endoplasmic reticulum proteins were only present in the sample that had been subjected to lipid extraction by incubating the homogenized cells in chloroform/methanol/water.     

'Information-Based-Acquisition' (IBA) technique with an ion-trap/time-of-flight mass spectrometer for high-throughput and reliable protein profiling

Highly complex protein mixtures can be analyzed after proteolysis using liquid chromatography/mass spectrometry (LC/MS). In an LC/MS run, intense peptide ions originating from high-abundance proteins are preferentially analyzed using tandem mass spectrometry (MS(2)), so obtaining the MS(2) spectra of peptide ions from low-abundance proteins is difficult even if such ions are detected. Furthermore, the MS(2) spectra may produce insufficient information to identify the peptides or proteins. To solve these problems, we have developed a real-time optimization technique for MS(2), called the Information-Based-Acquisition (IBA) system. In a preliminary LC/MS run, a few of the most intense ions detected in every MS spectrum are selected as precursors for MS(2) and their masses, charge states and retention times are automatically registered in an internal database. In the next run, a sample similar to that used in the first run is analyzed using database searching. Then, the ions registered in the database are excluded from the precursor ion selection to avoid duplicate MS(2) analyses. Furthermore, real-time de novo sequencing is performed just after obtaining the MS(2) spectrum, and an MS(3) spectrum is obtained for accurate peptide identification when the number of interpreted amino acids in the MS(2) spectrum is less than five. We applied the IBA system to a yeast cell lysate which is a typical crude sample, using a nanoLC/ion-trap time-of flight (IT/TOF) mass spectrometer, repeating the same LC/MS run five times. The obtained MS(2) and MS(3) spectra were analyzed by applying the Mascot (Matrix Science, Boston, MA, USA) search engine to identify proteins from the sequence database. The total number of identified proteins in five LC/MS runs was three times higher than that in the first run and the ion scores for peptide identification also significantly increased, by about 70%, when the MS(3) spectra were used, combined with the MS(2) spectra, before being subjected to Mascot analysis.     

Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.     

LC–MS/MS in the routine clinical laboratory: has its time come?

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been increasingly used in routine clinical laboratories during the last two decades. The high specificity, sensitivity, and multi-analyte potential make it an ideal alternative to immunoassays or conventional high-performance liquid chromatography (HPLC). It also provides higher throughput than gas chromatography–mass spectrometry (GC–MS). LC–MS/MS also offers higher flexibility than immunoassays because LC–MS/MS assays are typically developed in-house. In addition, abundant information can be obtained from a single LC–MS/MS run which can produce a large amount of quantitative or qualitative data. In this review, typical LC–MS/MS clinical applications are presented, personal experiences are shared, and strengths and weakness are discussed. It is foreseeable that LC–MS/MS will become a key instrument in routine clinical laboratories.     

Effect of mobile phase pH, aqueous-organic ratio, and buffer concentration on electrospray ionization tandem mass spectrometric fragmentation patterns: implications in liquid chromatography/tandem mass spectrometric bioanalysis

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) based on selected reaction monitoring (SRM) is the standard methodology in quantitative analysis of administered xenobiotics in biological samples. Utilizing two SRM channels during positive electrospray ionization (ESI) LC/MS/MS method development for a drug compound containing two basic functional groups, we found that the response ratio (SRM1/SRM2) obtained using an acidic mobile phase was dramatically different from that obtained using a basic mobile phase. This observation is different from the well-established phenomenon of mobile phase affecting the [M+H](+) response, which is directly related to the amount of the [M+H](+) ions produced during the ionization. Results from follow-up work reported herein revealed that the MS/MS fragmentation patterns of four drug or drug-like compounds are affected not only by the pH, but also by the aqueous-organic ratio of the mobile phase and the buffer concentration at a given apparent pH. The observed phenomenon can be explained by invoking that a mixture of [M+H](+) ions of the same m/z value for the analyte is produced that is composed of two or more species which differ only in the site of the proton attachment, which in turn affects their MS/MS fragmentation pattern. The ratio of the different protonated species changes depending on the pH, aqueous-organic ratio, or ionic strength of the mobile phase used. The awareness of the mobile phase dependency of the MS/MS fragmentation pattern of precursor ions of identical m/z value will influence LC/MS/MS-based bioanalytical method development strategies. Specifically, we are recommending that multiple SRM transitions be monitored during mobile phase screening, with the MS/MS parameters used for each SRM optimized for the composition of the mobile phase (pH, organic percentage, and ionic strength) in which the analyte elutes.     

Automated Precursor Ion Exclusion During LC-MS/MS Data Acquisition for Optimal Ion Identification

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used for characterizing multiple samples of complex mixtures with similar compositions. This article addresses a data acquisition strategy for collecting a maximal number of unique, high-quality MS/MS during LC-MS/MS analysis of multiple samples. Based on the concept that a component only needs to be identified once when analyzing multiple samples with similar compositions, an automated intersample data-dependent acquisition strategy was developed. The strategy is based on precursor ion exclusion (PIE) and is implemented in MassAnalyzer in an automated fashion for Thermo Scientific (San Jose, CA, USA) mass spectrometers. In this method, MassAnalyzer submits one sample at a time to the sample queue. After data acquisition of each sample, MassAnalyzer automatically analyzes the data to generate a PIE list based on the MS/MS precursor ions, merges this list with the list generated from previous runs, adds the list to the MS method file, and submits the next sample to the queue. The PIE list contains both m/z value and time window for each precursor ion, and is generated intelligently so that if an MS/MS is insufficient for identifying the peak of interest, it will be collected again near the top of the peak in the next run. Therefore, the strategy maximizes both quality and the number of unique MS/MS. When automated PIE was used to acquire LC-MS/MS data of an antibody tryptic digest and a soy hydrolysate sample, the number of identified ions increased by 52% and 93%, respectively, compared with data acquired without using PIE.     

High-sensitivity analysis of specific peptides in complex samples by selected MS/MS ion monitoring and linear ion trap mass spectrometry: application to biological studies

Mass spectrometry (MS) is a technique of paramount importance in Proteomics, and developments in this field have been possible owing to novel MS instrumentation, experimental strategies, and bioinformatics tools. Today it is possible to identify and determine relative expression levels of thousands of proteins in a biological system by MS analysis of peptides produced by proteolytic digestion. In some situations, however, the precise characterization of a particular peptide species in a very complex peptide mixture is needed. While single-fragment ion-based scanning modes such as selected ion reaction monitoring (SIRM) or consecutive reaction monitoring (CRM) may be highly sensitive, they do not produce MS/MS information and their actual specificity must be determined in advance, a prerequisite that is not usually met in a basic research context. In such cases, the MS detector may be programmed to perform continuous MS/MS spectra on the peptide ion of interest in order to obtain structural information. This selected MS/MS ion monitoring (SMIM) mode has a number of advantages that are fully exploited by MS detectors that, like the linear ion trap, are characterized by high scanning speeds. In this work, we show some applications of this technique in the context of biological studies. These results were obtained by selecting an appropriate combination of scans according to the purpose of each one of these research scenarios. They include highly specific identification of proteins present in low amounts, characterization and relative quantification of post-translational modifications such as phosphorylation and S-nitrosylation and species-specific peptide identification.     

Challenges in implementing clinical liquid chromatography‐tandem mass spectrometry methods ‐ seeing the light at the end of the tunnel

The use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) in the clinical setting is a relatively new application. One of the significant barriers hampering the transition of LC–MS/MS from the research lab into a clinical setting is the uncertainty of how to successfully develop and validate a method that meets guidelines for clinical applications. Here, we have taken this seemingly overwhelming process and broken it into five general stages for consideration: assessing the clinical validity of a new LC–MS/MS assay, determination of feasibility, assay development, assay validation and post‐implementation monitoring. Although various publications are available and serve as resources for determining development processes and acceptability criteria for specific LC–MS/MS assays, many of them are general recommendations or are specific to research applications that may not translate either practically or clinically. In this perspective special feature article, a resource is compiled that describes key differences between LC–MS/MS methods for research use versus clinical use. In addition, the challenges facing the expanding role of this technique in the clinical setting are discussed, including instrumentation/automation challenges, potential regulation of laboratory developed tests by the US Food and Drug Administration and standardization and harmonization of MS methods through the use of traceable materials and availability of guidance documents. Copyright © 2013 John Wiley & Sons, Ltd.     

钼催化剂上甲醇直接气相羰基化活性与吸附的关系

 制备了一系列非负载的钼催化剂，考察了催化剂上甲醇直接气相\r\n羰化活性与反应物及产物吸附的关系．结果发现，催化剂的制备方法对\r\n甲醇直接气相羰化活性的影响很明显，以纯二硫化钼试剂制备的催化剂\r\n，甲醇转化率与羰化产物乙酸甲酯的选择性最低；以三氧化钼气相还原\r\n硫化制备的催化剂，其羰化活性较低；以钼酸铵溶液经硫化铵溶液或硫\r\n化氢气体硫化制得的催化剂，其性能明显优于前两种，尤以钼酸铵经硫\r\n化铵溶液硫化制得的催化剂的性能最佳．催化剂的羰化活性明显依赖于\r\n催化剂的吸附性能，羰化活性高的催化剂不仅吸附CO的量大，而且对甲\r\n醇和乙酸甲酯的吸附量也明显较大．     

Development of algorithms for automated elucidation of spectral feature/substructure relationships in tandem mass spectrometry

A pattern-recognition/artificial-intelligence program, referred to as MAPS (method for analyzing patterns in spectra), is described for the identification of relationships that exist between the presence of substructures in molecules and the characteristic features they produce in mass spectrometry (MS) and MS/MS data. The MAPS algorithm discovers these relationships by intelligent analysis of a data base of MS and MS/MS spectra. The relationships found are expressed as rules, which may then be used to identify characterized substructures in “unknowns”. No prior knowledge of fragmentation pathways or rearrangements is assumed in the rule-generation process. While MAPS currently uses MS and MS/MS data, the approach (and much of the software) is equally suited to multiple-stage mass spectrometric data.     

Ultrafast liquid chromatography/ultraviolet and liquid chromatography/tandem mass spectrometric analysis

Optimal liquid chromatography/mass spectrometric [LC/MS(/MS)] analysis depends on both the LC selectivity and the electrospray efficiency. Here, we outline a simple and comprehensive LC/MS/MS strategy for the rapid analysis of a wide range of pharmaceutical compounds. To achieve ultrafast LC separation with little sacrifice in peak capacity, one needs to start with a column that provides a good peak capacity at short gradient run times; secondly, it is important to use high flow rates to achieve a good gradient peak capacity. Following this strategy, it was possible to baseline-resolve a mixture (containing acidic, neutral, and basic pharmaceutical analytes) in seconds. By coupling the selectivity provided by fast LC separation with the specificity of MS/MS detection, it is possible to separate and identify a wide range of analytes in 1-min gradient analyses. Also, the impact of mobile phase pH on both the chromatographic selectivity and the MS/MS sensitivity is demonstrated.     

Cutting-edge LC–MS/MS applications in clinical mass spectrometry: Focusing on analysis of drugs and metabolites

In recent years, liquid chromatography with tandem mass spectrometry (LC–MS/MS) has become a fundamental technology in clinical practice. In Japan, the LC–MS/MS system is used in many large hospitals. It has become popular among pharmacists and laboratory technicians. LC–MS/MS has some advantages in terms of accuracy, speed, and comprehensiveness compared to conventional automated chemical testing equipment. However, LC–MS/MS is by no means a universal method, and it is necessary to understand its characteristics before using it. In the field of therapeutic drug monitoring (TDM), there is an issue with linearity in comprehensive measurement; however, ion-abundance adjustment methods, such as in-source collision-induced dissociation, have been proposed as a solution to this problem. The development of a biomarker analysis includes search, identification, and quantification, and it is necessary to select an appropriate mass spectrometric method for each step. In this paper, we review cutting-edge technologies that can expand the performance of LC–MS/MS in the clinical field and consider current issues and future prospects.     

快速溶剂萃取-气相色谱/质谱联用测定塑料桌布中的邻苯二甲酸酯

采用快速溶剂萃取-气相色谱/质谱联用法(ASE-GC/MS)和超声波萃取-气相色谱/质谱联用法(USE-GC/MS)测定了塑料桌布中的邻苯二甲酸酯.结果表明:ASE-GC/MS的测量精度优于USE-GC/MS,后者测量值大约为前者的五分之四;ASE-GC/MS的回收率为89.0%～95.5%,相对标准偏差为4.3%～1...     

The development and assessment of high‐throughput mass spectrometry‐based methods for the quantification of a nanoparticle drug delivery agent in cellular lysate

The safe use of lipid‐based drug delivery agents requires fast and sensitive qualitative and quantitative assessment of their cellular interactions. Many mass spectrometry (MS) based analytical platforms can achieve such task with varying capabilities. Therefore, four novel high‐throughput MS‐based quantitative methods were evaluated for the analysis of a small organic gene delivery agent: N,N‐bis(dimethylhexadecyl)‐1,3‐propane‐diammonium dibromide (G16‐3). Analysis utilized MS instruments that detect analytes using low‐resolution tandem MS (MS/MS) analysis (i.e. QTRAP or linear ion trap in this work) or high‐resolution MS analysis (i.e. time of flight (ToF) or Orbitrap). Our results indicate that the validated fast chromatography (FC)‐QTRAP‐MS/MS, FC‐ LTQ‐Orbitrap‐MS, desorption electrospray ionization‐collision‐induced dissociation (CID)‐MS/MS and matrix assisted laser desorption ionization‐ToF/ToF‐MS MS methods were superior in the area of method development and sample analysis time to a previously developed liquid chromatography (LC)‐CID‐MS/MS. To our knowledge, this is the first evaluation of the abilities of five MS‐based quantitative methods that target a single pharmaceutical analyte. Our findings indicate that, in comparison to conventional LC‐CID‐MS/MS, the new MS‐based methods resulted in a (1) substantial reduction in the analysis time, (2) reduction in the time required for method development and (3) production of either superior or comparable quantitative data. The four new high‐throughput MS methods, therefore, were faster, more efficient and less expensive than a conventional LC‐CID‐MS/MS for the quantification of the G16‐3 analyte within tissue culture. When applied to cellular lysate, no significant change in the concentration of G16‐3 gemini surfactant within PAM212 cells was observed between 5 and 53 h, suggesting the absence of any metabolism/excretion from PAM212 cells. Copyright © 2014 John Wiley & Sons, Ltd.     

High-throughput quantitative bioanalysis by LC/MS/MS

This review article discusses the most recent significant advances in the sample preparation and mass spectrometry aspects of high-throughput bioanalysis by LC/MS/MS for the quantitation of drugs, metabolites and endogenous biomolecules in biological matrices. The introduction and implementation of automated 96-well extraction has brought about high-throughput approaches to the biological sample preparation techniques of solid-phase extraction, liquid-liquid extraction and protein precipitation. The fast-flow on-line extraction technique is a different high-throughput approach that has also significantly speeded up analysis by LC/MS/MS. The use of pierceable caps for biological tubes further enhances the analysis speed and improves the safety in handling biological samples. The need for adequate chromatographic separation in order to eliminate interferences due to metabolites and/or matrix effects in LC/MS/MS is discussed. To highlight our limited understanding of atmospheric pressure ionization mass spectrometry, results from recent investigations that appear to be counter-intuitive are presented. Looking ahead to the future, multiplexed LC/MS/MS systems and capillary LC are presented as areas that can bring about further improvements in analysis speed and sensitivity to quantitative bioanalysis by LC/MS/MS.