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Elevating the column temperature is an effective strategy for improving the chromatographic separation of peptides. However, high temperatures induce artificial modifications that compromise the quality of the peptide analysis. Here, we present a novel high-temperature LC-MS method that retains the benefits of a high column temperature while significantly reducing peptide modification and degradation during reversed-phase liquid chromatography. Our approach leverages a short inline trap column maintained at a near-ambient temperature installed upstream of a separation column. The retentivity and dimensions of the trap column were optimized to shorten the residence time of peptides in the heated separation column without compromising the separation performance. This easy-to-implement approach increased peak capacity by 1.4-fold within a 110 min peptide mapping of trastuzumab and provided 10% more peptide identifications in exploratory LC-MS proteomic analyses compared with analyses conducted at 30 °C while maintaining the extent of modifications close to the background level. In the peptide mapping of biopharmaceuticals, where in-column modifications can falsely elevate the levels of some critical quality attributes, the method reduced temperature-related artifacts by 66% for N-terminal pyroGlu and 63% for oxidized Met compared to direct injection at 60 °C, thus improving reliability in quality control of protein drugs. Our findings represent a promising advancement in LC-MS methodology, providing researchers and industry professionals with a valuable tool for improving the chromatographic separation of peptides while significantly reducing the unwanted modifications.

Data-independent acquisition (DIA) has improved the identification and quantitation coverage of peptides and proteins in liquid chromatography-tandem mass spectrometry-based proteomics. However, different DIA data-processing tools can produce very different identification and quantitation results for the same data set. Currently, benchmarking studies of DIA tools are predominantly focused on comparing the identification results, while the quantitative accuracy of DIA measurements is acknowledged to be important but insufficiently investigated, and the absence of suitable metrics for comparing quantitative accuracy is one of the reasons. A new metric is proposed for the evaluation of quantitative accuracy to avoid the influence of differences in false discovery rate control stringency. The part of the quantitation results with high reliability was acquired from each DIA tool first, and the quantitative accuracy was evaluated by comparing quantification error rates at the same number of accurate ratios. From the results of four benchmark data sets, the proposed metric was shown to be more sensitive to discriminating the quantitative performance of DIA tools. Moreover, the DIA tools with advantages in quantitative accuracy were consistently revealed by this metric. The proposed metric can also help researchers in optimizing algorithms of the same DIA tool and sample preprocessing methods to enhance quantitative accuracy.

Growing clinical evidence reveals that systematic molecular alterations in the brain occur 20 years before the onset of AD pathological features. Apolipoprotein E4 (ApoE4) is one of the most significant genetic risk factors for Alzheimer's disease (AD), which is not only associated with the AD pathological features such as amyloid-β deposition, phosphorylation of tau proteins, and neuroinflammation but is also involved in metabolism, neuron growth, and synaptic plasticity. Multiomics, such as metabolomics and proteomics, are applied widely in identifying key disease-related molecular alterations and disease-progression-related changes. Despite recent advances in the development of analytical technologies, screening the entire profile of metabolites remains challenging due to the numerous classes of compounds with diverse chemical properties that require different extraction processes for mass spectrometry. In this study, we utilized Orbitrap Secondary Ion Mass Spectrometry (OrbiSIMS) as a chemical filtering screening tool to examine molecular alterations in ApoE4-carried neuroglioma cells compared to wild-type H4 cells. The findings were compared using liquid chromatography (LC)-MS/MS targeted metabolomics analysis for the confirmation of specific metabolite classes. Detected alterations in peptide fragments by OrbiSIMS provided preliminary indications of protein changes. These were extensively analyzed through proteomics to explore ApoE4's impact on proteins. Our metabolomics approach, combining OrbiSIMS and LC-MS/MS, revealed disruptions in lipid metabolism, including glycerophospholipids and sphingolipids, as well as amino acid metabolism, encompassing alanine, aspartate, and glutamate metabolism; aminoacyl-tRNA biosynthesis; glutamine metabolism; and taurine and hypotaurine metabolism. Further LC-MS/MS proteomics studies confirmed the dysfunction in amino acid and tRNA aminoacylation metabolic processes, and highlighted RNA splicing alterations influenced by ApoE4.

This Technical Note describes a dual-column liquid chromatography system coupled to mass spectrometry (LC-MS) for high-throughput bottom-up proteomic analysis. This system made full use of two 2-position 10-port valves and a binary pump with an integrated loading pump of a commercial LC instrument to provide successive operation of two parallel subsystems. Each subsystem consisted of a set of trap columns and an analytical column. A T-junction union was used to split the mobile phase from the loading pump into two parts. This allowed one set of columns to be washed and equilibrated, followed by the injection of the next sample, while the previous sample was eluting and being analyzed on the other set of columns, thereby greatly increasing the analysis throughput. This approach showed high reproducibility for the analysis of HeLa tryptic digests with average relative standard deviation (RSD) values of 1.75%, 6.90%, and 5.19% for the identification number of proteins, peptides, and peptide-spectrum matches (PSMs), respectively, across 10 consecutive runs. The capacity for peptide and protein identification, as well as proteome depth, of the dual-column LC system was comparable to a conventional single-column system. Due to its simple equipment requirements and set up process, this method should be highly accessible for other laboratories.

This work demonstrates the utility of high-throughput nanoLC-MS and label-free quantification (LFQ) for sample-limited bottom-up proteomics analysis, including single-cell proteomics (SCP). Conditions were optimized on a 50 μm internal diameter (I.D.) column operated at 100 nL/min in the direct injection workflow to balance method sensitivity and sample throughput from 24 to 72 samples/day. Multiple data acquisition strategies were also evaluated for proteome coverage, including data-dependent acquisition (DDA), wide-window acquisition (WWA), and wide-window data-independent acquisition (WW-DIA). Analyzing 250 pg HeLa digest with a 10-min LC gradient (72 samples/day) provided >900, >1,800, and >3,000 protein group identifications for DDA, WWA, and WW-DIA, respectively. Total method cycle time was further reduced from 20 to 14.4 min (100 samples/day) by employing a trap-and-elute workflow, enabling 70% mass spectrometer utilization. The method was applied to library-free DIA analysis of single-cell samples, yielding >1,700 protein groups identified. In conclusion, this study provides a high-sensitivity, high-throughput nanoLC-MS configuration for sample-limited proteomics.

Due to its sensitivity and productivity, bottom-up proteomics based on liquid chromatography-mass spectrometry (LC-MS) has become the core approach in the field. The de facto standard LC-MS platform for proteomics operates at sub-µL/min flow rates, and nanospray is required for efficiently introducing peptides into a mass spectrometer. Although this is almost a "dogma", this view is being reconsidered in light of developments in highly efficient chromatographic columns, and especially with the introduction of exceptionally sensitive MS instruments. Although conventional-flow LC-MS platforms have recently penetrated targeted proteomics successfully, their possibilities in discovery-oriented proteomics have not yet been thoroughly explored. Our objective was to determine what are the extra costs and what optimization and adjustments to a conventional-flow LC-MS system must be undertaken to identify a comparable number of proteins as can be identified on a nanoLC-MS system. We demonstrate that the amount of a complex tryptic digest needed for comparable proteome coverage can be roughly 5-fold greater, providing the column dimensions are properly chosen, extra-column peak dispersion is minimized, column temperature and flow rate are set to levels appropriate for peptide separation, and the composition of mobile phases is fine-tuned. Indeed, we identified 2 835 proteins from 2 µg of HeLa cells tryptic digest separated during a 60 min gradient at 68 µL/min on a 1.0 mm x 250 mm column held at 55 °C and using an aqua-acetonitrile mobile phases containing 0.1% formic acid, 0.4% acetic acid, and 3% dimethyl sulfoxide. Our results document that conventional-flow LC-MS is an attractive alternative for bottom-up exploratory proteomics. [ABSTRACT FROM AUTHOR]

For LC-MS-based targeted quantification of biotherapeutics and biomarkers in clinical and pharmaceutical environments, high sensitivity, high throughput, and excellent robustness are all essential but remain challenging. For example, though nano-LC-MS has been employed to enhance analytical sensitivity, it falls short because of its low loading capacity, poor throughput, and low operational robustness. Furthermore, high chemical noise in protein bioanalysis typically limits the sensitivity. Here we describe a novel trapping-micro-LC-MS (T-μLC-MS) strategy for targeted protein bioanalysis, which achieves high sensitivity with exceptional robustness and high throughput. A rapid, high-capacity trapping of biological samples is followed by μLC-MS analysis; dynamic sample trapping and cleanup are performed using pH, column chemistry, and fluid mechanics separate from the μLC-MS analysis, enabling orthogonality, which contributes to the reduction of chemical noise and thus results in improved sensitivity. Typically, the selective-trapping and -delivery approach strategically removes >85% of the matrix peptides and detrimental components, markedly enhancing sensitivity, throughput, and operational robustness, and narrow-window-isolation selected-reaction monitoring further improves the signal-to-noise ratio. In addition, unique LC-hardware setups and flow approaches eliminate gradient shock and achieve effective peak compression, enabling highly sensitive analyses of plasma or tissue samples without band broadening. In this study, the quantification of 10 biotherapeutics and biomarkers in plasma and tissues was employed for method development. As observed, a significant sensitivity gain (up to 25-fold) compared with that of conventional LC-MS was achieved, although the average run time was only 8 min/sample. No appreciable peak deterioration or loss of sensitivity was observed after >1500 injections of tissue and plasma samples. The developed method enabled, for the first time, ultrasensitive LC-MS quantification of low levels of a monoclonal antibody and antigen in a tumor and cardiac troponin I in plasma after brief cardiac ischemia. This strategy is valuable when highly sensitive protein quantification in large sample sets is required, as is often the case in typical biomarker validation and pharmaceutical investigations of antibody therapeutics. [ABSTRACT FROM AUTHOR]

Quantitative cross-linking/mass spectrometry (QCLMS) is an emerging approach to study conformational changes of proteins and multi-subunit complexes. Distinguishing protein conformations requires reproducibly identifying and quantifying cross-linked peptides. Here we analyzed the variation between multiple cross-linking reactions using bis[sulfosuccinimidyl] suberate (BS3)-cross-linked human serum albumin (HSA) and evaluated how reproducible cross-linked peptides can be identified and quantified by LC-MS analysis. To make QCLMS accessible to a broader research community, we developed a workflow that integrates the established software tools MaxQuant for spectra preprocessing, Xi for cross-linked peptide identification, and finally Skyline for quantification (MS1 filtering). Out of the 221 unique residue pairs identified in our sample, 124 were subsequently quantified across 10 analyses with coefficient of variation (CV) values of 14% (injection replica) and 32% (reaction replica). Thus our results demonstrate that the reproducibility of QCLMS is in line with the reproducibility of general quantitative proteomics and we establish a robust workflow for MS1-based quantitation of cross-linked peptides.ᅟ [ABSTRACT FROM AUTHOR]

Glycated proteins are emerging as good indicators for diabetes and age related diseases. However, the platform for analysis of glycated proteome has been relatively less well established. We here introduce an online 2D-LC-HCD-MS/MS platform for comprehensive glycated peptide quantification. This platform includes a boronate affinity column in the first dimension for enrichment, reversed phase nanoLC column in the second dimension for separation, a benchtop Orbitrap mass spectrometer with HCD-MS/MS for peptide sequencing, and MaxQuant bioinformatics tool for identification and quantification of glycated peptides. This online 2D-LC-HCD-MS/MS platform has high enrichment efficiency with 85% of identified peptides in the enriched fraction as glycated, high sensitivity for detection of glycated peptides with LOD and LOQ at 1.2 and 2.4 pg, respectively, and high reproducibility with interday CVs < 20% for 80% of the glycated peptides. The number of glycated peptides quantified in in vitro glycated human plasma increased more than 3-fold using this platform in comparison to that obtained using 1D-LC-HCD-MS/MS platform without boronate affinity enrichment. Application of this online platform to human plasma identified 376 glycated peptides from 10 μg of protein digests. This highly sensitive and reproducible online 2D platform is promising for glycated protein analysis of complex clinical samples. [ABSTRACT FROM AUTHOR]

With recent advances in understanding the genomic underpinnings and oncogenic drivers of pathogenesis in different subtypes, it is increasingly clear that proper pretreatment diagnostics are essential for the choice of appropriate treatment options for non-small cell lung cancer (NSCLC). Tumor tissue preservation in optimal cutting temperature (OCT) compound is commonly used in the surgical suite. However, proteins recovered from OCT-embedded specimens pose a challenge for LC-MS/MS experiments, due to the large amounts of polymers present in OCT. Here we present a simple workflow for whole proteome analysis of OCT-embedded NSCLC tissue samples, which involves a simple trichloroacetic acid precipitation step. Comparisons of protein recovery between frozen versus OCT-embedded tissue showed excellent consistency with more than 9200 proteins identified. Using an isobaric labeling strategy, we quantified more than 5400 proteins in tumor versus normal OCT-embedded core needle biopsy samples. Gene ontology analysis indicated that a number of proliferative as well as squamous cell carcinoma (SqCC) marker proteins were overexpressed in the tumor, consistent with the patient's pathology based diagnosis of 'poorly differentiated SqCC'. Among the most downregulated proteins in the tumor sample, we noted a number of proteins with potential immunomodulatory functions. Finally, interrogation of the aberrantly expressed proteins using a candidate approach and cross-referencing with publicly available databases led to the identification of potential druggable targets in DNA replication and DNA damage repair pathways. We conclude that our approach allows LC-MS/MS proteomic analyses on OCT-embedded lung cancer specimens, opening the way to bring powerful proteomics into the clinic. [ABSTRACT FROM AUTHOR]

Glycomics and glycoproteomics analyses by mass spectrometry require efficient front-end separation methods to enable deep characterization of heterogeneous glycoform populations. Chromatography methods are generally limited in their ability to resolve glycoforms using mobile phases that are compatible with online liquid chromatography-mass spectrometry (LC-MS). The adoption of capillary electrophoresis-mass spectrometry methods (CE-MS) for glycomics and glycoproteomics is limited by the lack of convenient interfaces for coupling the CE devices to mass spectrometers. Here, we describe the application of a microfluidics-based CE-MS system for analysis of released glycans, glycopeptides and monosaccharides. We demonstrate a single CE method for three different modalities, thus contributing to comprehensive glycoproteomics analyses. In addition, we explored compatible sample derivatization methods. We used glycan TMT-labeling to improve electrophoretic migration and enable multiplexed quantitation by tandem MS. We used sialic acid linkage-specific derivatization methods to improve separation and the level of information obtained from a single analytical step. Capillary electrophoresis greatly improved glycoform separation for both released glycans and glycopeptides over that reported for chromatography modes more frequently employed for such analyses. Overall, the CE-MS method described here enables rapid setup and analysis of glycans and glycopeptides using mass spectrometry. [ABSTRACT FROM AUTHOR]

We present a novel absorption correction approach for elemental distribution images obtained with a laboratory confocal micro X-ray fluorescence spectrometer. The procedure is suited especially for biological samples, as a constant dark matrix with a varying minor or trace element distribution is assumed. The constant absorption in the sample is extracted from depth dependent measurements. By using the concept of an effective excitation energy, depth-dependent, and element-specific excitation energy values are calculated. For each voxel of a full 3D measurement, a correction is performed taking into account the actual number of voxels in the excitation and detection path. As proof of concept, the embryonic region of pearl millet seeds is investigated. Data are measured from the top and bottom side, resulting in a good agreement after the application of the absorption correction procedure. The distribution of elemental micronutrients is compared in seeds of two pearl millet genotypes. The corrected images illustrate different localization patterns of the micronutrient elements in pearl millet seed tissues. [ABSTRACT FROM AUTHOR]

Protein arginine methylation is one of the common post-translational modifications in cellular processes. To date, two isomeric forms of dimethylated arginine have been identified: asymmetric NG,NG-dimethylarginine (aDMA), and symmetric NG,N'G-dimethylarginine (sDMA). Evidence indicated that these isomers can coexist and have different or even opposite functions, with aDMA and sDMA forms of arginine 2 on histone H3 (i.e., H3R2me2a and H3R2me2s) being an example. Thus, specific detection and quantification of each isomeric form is important. Current methods are capable of predicting and detecting thousands of methylarginine sites in proteins, whereas differentiation and stoichiometric measurement of dimethylated protein isomers are still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics has emerged as a promising technique for site-specific quantification of protein methylation using enzymatic peptides as surrogates of target proteins. However, it should be pointed out that a routine targeted proteomics strategy cannot easily distinguish sDMA- and aDMA-containing surrogate peptides due to their common nature. The estimated amount should be considered as the sum of both arginine dimethylated isomers. In this study, compositional analysis based on a linear algebra algorithm as an add-on to targeted proteomics was employed to quantify H3R2me2a and H3R2me2s (i.e., surrogate peptides of AR(me2a)TK(me1/2)QT and AR(me2s)TK(me1/2)QT). To achieve this simultaneous quantification, a targeted proteomics assay was developed and validated for each isomer first. With the slope and intercept of their calibration curves for each multiple reaction monitoring (MRM) transition, linear algebraic equations were derived. Using a series of mock mixtures consisting of isomers in varying concentrations, the reliability of the method was confirmed. Finally, the H3R2 dimethylation status was analyzed in normal MCF-10A cells, parental drug-sensitive MCF-7/WT cancer cells, and drug-resistant MCF-7/ADR cancer cells. Dimethylated H3R2 was also monitored in MCF-7/WT cells with the treatment of doxorubicin (DOX) for confirmation. [ABSTRACT FROM AUTHOR]

Protein structural and functional studies rely on complete qualitative and quantitative information on protein species (proteoforms); thus, it is important to quantify differentially expressed proteins at their molecular level. Here we report our development of universal pseudoisobaric dimethyl labeling (pIDL) of amino groups at both the N-terminal and lysine residues for relative quantitation of intact proteins. Initial proof-of-principle study was conducted on standard protein myoglobin and hepatocellular proteomes (HepG2 vs LO2). The amino groups from both the N-terminal and lysine were dimethylated with HXHO (X = 13C or C) and NaBY3CN (Y = H or D). At the standard protein level, labeling efficiency, effect of product ion size, and mass resolution on quantitation accuracy were explored; and a good linear quantitation dynamic range up to 50-fold was obtained. For the hepatocellular proteome samples, 33 proteins were quantified with RSD = 10% from one-dimensional reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) analysis of the 1:1 mixed samples. The method in this study can be extended to quantitation of other intact proteome systems. The universal "one-pot" dimethyl labeling of all the amino groups in a protein without the need of preblocking of those on the lysine residues is made possible by protein identification and quantitation analysis using ProteinGoggle 2.0 with customized databases of both precursor and product ions containing heavy isotopes. [ABSTRACT FROM AUTHOR]

We present a novel proteomic standard for assessing liquid chromatography--tandem mass spectrometry (LC-MS/MS) instrument performance, in terms of chromatographic reproducibility and dynamic range within a single LC-MS/MS injection. The peptide mixture standard consists of six peptides that were specifically synthesized to cover a wide range of hydrophobicities (grand average hydropathy (GRAVY) scores of -0.6 to 1.9). A combination of stable isotope labeled amino adds (13C and 15N) were inserted to create five isotopologues. By combining these isotopologues at different ratios, they span four orders of magnitude within each distinct peptide sequence. Each peptide, from lightest to heaviest, increases in abundance by a factor of 10. We evaluate several metrics on our quadrupole orbitrap instrument using the 6 x 5 LC-MS/MS reference mixture spiked into a complex lysate background as a function of dynamic range, including mass measurement accuracy (MMA) and the linear range of quantitation of MSI and parallel reaction monitoring experiments. Detection and linearity of the instrument routinely spanned three orders of magnitude across the gradient (500 frnol to 0.5 frnol on column) and no systematic trend was observed for MMA of targeted peptides as a function of abundance by analysis of variance analysis (p = 0.17). Detection and linearity of the fifth isotopologue (i.e., 0.05 frnol on column) was dependent on the peptide and instrument scan type (MSI vs PRM). We foresee that this standard will serve as a powerful method to conduct both intra-instrument performance monitoring/evaluation, technology development, and inter-instrument comparisons. [ABSTRACT FROM AUTHOR]

We describe a proteomic reactor-based homogeneous phase enrichment of cysteine-containing peptides in a filter aided sample preparation (FASP) format. In this approach thiol-reduced proteins are derivatized with thiol-activated polyethylene glycol (TAPEG) before protein cleavage. Consecutive digestion with endoproteinase LysC and trypsin allows isolation of twofractions of nonderivatized peptides. After reduction of disulfide bonds between cysteine-containing peptides and the polyethylene glycol moieties, a third fraction of peptides is collected. LC-MS/MS analyses revealed that on average this fraction consists of 95% cysteine-containing peptides. Since 85-93% of all peptides are unique to a single subfraction, the combination of TAPEG and FASP offers an efficient peptide separation strategy. Analysis of whole cell lysates of mouse brain, liver, red muscle fibers, and CaCo-2 cells using the TAPEG FASP approach allowed identification of 6,900, 5,800, 4,200 and 7,900 proteins, 10-30% more than were identified using two-step digestion without isolation of Cys-containing peptides. The fractionation also increased the protein sequence coverage by 10-30%. [ABSTRACT FROM AUTHOR]

In the past decade, several strategies for comprehensive phosphoproteome analysis have been introduced. Most of them combine different phosphopeptide enrichment techniques and require starting material in the milligram range, as a consequence of their insufficient sensitivity. This limitation impairs the applicability of phosphoproteomics to a wide variety of clinical research, where sample material is highly limited. Here we introduce a highly sensitive and easy-to-establish 2D bottom-up strategy for microgram-scale phosphoproteomics, based on electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), a simple solid-phase extraction step by strong cation exchange (SCX) or reversed phase (RP), and LC-MS analysis. With only 100 µg of tryptic digested, nonstimulated HeLa protein and 45 h of LC-MS analysis time, we identified ≥7500 nonredundant and highly confident phosphorylation sites (per replicate). We assigned all phosphorylation sites to 3013 phosphoproteins, covering the entire dynamic range from 107 down to a few copies per cell. Compared to affinity-based-enrichment methods using Ti4+, our ERLIC-based strategy enriched considerably longer and more acidic phosphopeptides and consequently, we identified 327 phosphorylated C-terminal peptides. The simplicity and high sensitivity of ERLIC-SCX/RP-LC-MS render its future promising for microgram-scale-phosphoproteomics in biological, biomedical, and clinical research. [ABSTRACT FROM AUTHOR]

This study presents on-tissue proteolytic digestion using a microwave irradiation and peptide extraction method for in situ analysis of proteins from spatially defined regions of a tissue section. The methodology utilizes hydrogel discs (1 mm diameter) embedded with trypsin solution. The enzyme-laced hydrogel discs are applied to a tissue section, directing enzymatic digestion to a spatially confined area of the tissue. By applying microwave radiation, protein digestion is performed in 2 min on-tissue, and the extracted peptides are then analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). The reliability and reproducibility of the microwave assisted hydrogel mediated on-tissue digestion is demonstrated by the comparison with other on-tissue digestion strategies, including comparisons with conventional heating and in-solution digestion. LC-MS/MS data were evaluated considering the number of identified proteins as well as the number of protein groups and distinct peptides. The results of this study demonstrate that rapid and reliable protein digestion can be performed on a single thin tissue section while preserving the relationship between the molecular information obtained and the tissue architecture, and the resulting peptides can be extracted in sufficient abundance to permit analysis using LC-MS/MS. This approach will be most useful for samples that have limited availability but are needed for multiple analyses, especially for the correlation of proteomics data with histology and immunohistochemistry. [ABSTRACT FROM AUTHOR]

Evidence will be presented that in the article "Novel LC-MS2 Product Dependent Parallel Data Acquisition Function and Data Analysis Workflow for Sequencing and Identification of Intact Glycopeptides" written by Wu, S.-W.; Pu, T.-H.; Viner, R.; Khoo, K.-H. in Anal. Chem. 2014, 86, 5478-5486, noncovalent homo- and heterodimers were mis-identified as glycopeptides bearing well-defined N-linked structures, where the unexplained mass was attributed to excessive O-glycosylation. Noncovalent dimer formation of abundant components has not previously been considered as a complication in high-throughput proteomic analyses. [ABSTRACT FROM AUTHOR]

Quantitative metabolomics and proteomics technologies are powerful approaches to explore cellular metabolic regulation. Unfortunately, combining the two technologies typically requires different LC-MS setups for sensitive measurement of metabolites and peptides. One approach to enhance the analysis of certain classes of metabolites is by derivatization with various types of tags to increase ionization and chromatographic efficiency. We demonstrate here that derivatization of amine metabolites with tandem mass tags (TMT), typically used in multiplexed peptide quantitation, facilitates amino acid analysis by standard nanoflow reversed-phase LC-MS setups used for proteomics. We demonstrate that this approach offers the potential to perform experiments at the MSl-level using duplex tags or at the MS2-Ievel using novel 10-plex reporter ion-containing isobaric tags for multiplexed amine metabolite analysis. We also demonstrate absolute quantitative measurements of amino acids conducted in parallel with multiplexed quantitative proteomics, using similar LC-MS setups to explore cellular amino acid regulation. We further show that the approach can also be used to determine intracellular metabolic labeling of amino acids from glucose carbons. [ABSTRACT FROM AUTHOR]

Statistical process control (SPC) is a robust set of tools that aids in the visualization, detection, and identification of assignable causes of variation in any process that creates products, services, or information. A tool has been developed termed Statistical Process Control in Proteomics (SProCoP) which implements aspects of SPC (e.g., control charts and Pareto analysis) into the Skyline proteomics software. It monitors five quality control metrics in a shotgun or targeted proteomic workflow. None of these metrics require peptide identification. The source code, written in the R statistical language, runs directly from the Skyline interface, which supports the use of raw data files from several of the mass spectrometry vendors. It provides real time evaluation of the chromatographic performance (e.g., retention time reproducibility, peak asymmetry, and resolution), and mass spectrometric performance (targeted peptide ion intensity and mass measurement accuracy for high resolving power instruments) via control charts. Thresholds are experiment- and instrument-specific and are determined empirically from user-defined quality control standards that enable the separation of random noise and systematic error. Finally, Pareto analysis provides a summary of performance metrics and guides the user to metrics with high variance. The utility of these charts to evaluate proteomic experiments is illustrated in two case studies. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Shotgun proteomics experiments integrate a complex sequence of processes, any of which can introduce variability. Quality metrics computed from LC-MS/MS data have relied upon identifying MS/MS scans, but a new mode for the QuaMeter software produces metrics that are independent of identifications. Rather than evaluating each metric independently, we have created a robust multivariate statistical toolkit that accommodates the correlation structure of these metrics and allows for hierarchical relationships among data sets. The framework enables visualization and structural assessment of variability. Study 1 for the Clinical Proteomics Technology Assessment for Cancer (CPTAC), which analyzed three replicates of two common samples at each of two time points among 23 mass spectrometers in nine laboratories, provided the data to demonstrate this framework, and CPTAC Study 5 provided data from complex lysates under Standard Operating Procedures (SOPs) to complement these findings. Identification-independent quality metrics enabled the differentiation of sites and run-times through robust principal components analysis and subsequent factor analysis. Dissimilarity metrics revealed outliers in performance, and a nested ANOVA model revealed the extent to which all metrics or individual metrics were impacted by mass spectrometer and run time. Study 5 data revealed that even when SOPs have been applied, instrument-dependent variability remains prominent, although it may be reduced, while within-site variability is reduced significantly. Finally, identification-independent quality metrics were shown to be predictive of identification sensitivity in these data sets. QuaMeter and the associated multivariate framework are available from http://fenchurch.mc.vanderbilt.edu and http://homepages.uc.edu/~wang2x7/, respectively. [ABSTRACT FROM AUTHOR]

Accurate quantification is a fundamental requirement in the fields of proteomics and biomarker discovery, and for clinical diagnostic assays. To demonstrate the extent of quantitative variability in measurable peptide concentrations due to differences among "typical" protein digestion protocols, the model protein, human serum albumin (HSA), was subjected to enzymatic digestion using 12 different sample preparation methods, and separately, was examined through a comprehensive timecourse of trypsinolysis. A variety of digestion conditions were explored including differences in digestion time, denaturant, source of enzyme, sample cleanup, and denaturation temperature, among others. Timecourse experiments compared differences in relative peptide concentrations for tryptic digestions ranging from 15 min to 48 h. A predigested stable isotope-labeled (15N) form of the full-length (HSA) protein, expressed in yeast was spiked into all samples prior to LC-MS analysis to compare yields of numerous varieties of tryptic peptides. Relative quantification was achieved by normalization of integrated extracted ion chromatograms (XICs) using liquid chromatography-tandem mass spectrometry (LCMS/ MS) by multiple-reaction monitoring (MRM) on a triple quadrupole (QQQ) MS. Related peptide fragmentation transitions, and multiple peptide charge states, were monitored for validation of quantitative results. Results demonstrate that protein concentration was shown to be unequal to tryptic peptide concentrations for most peptides, including so-called "proteotypic" peptides. Peptide release during digestion displayed complex kinetics dependent on digestion conditions and, by inference, from denatured protein structure. Hydrolysis rates at tryptic cleavage sites were also shown to be affected by differences in nearest and next-nearest amino acid residues. The data suggesting nonstoichiometry of enzymatic protein digestions emphasizes the often overlooked difficulties for routine absolute protein quantification, and highlights the need for use of suitable internal standards and isotope dilution techniques. [ABSTRACT FROM AUTHOR]

The gel-based proteomic analysis of plasma membranes from rat liver and chemically induced, malignant hepatocellular carcinoma Morris hepatoma 7777 was systematically optimized to yield the maximum number of proteins containing transmembrane domains (TMDs). Incorporation of plasma membrane proteins into a polyacrylamide "tube gel" followed by in-gel digestion of "tube gel" pieces significantly improved detection by electrospray ionization-liquid chromatography-tandem mass spectrometry. Removal of less hydrophobic proteins by washing isolated plasma membranes with 0.1 M sodium carbonate enables detection of a higher number of hydrophobic proteins containing TMDs in both tissues. Subsequent treatment of plasma membranes by a proteolytic enzyme (trypsin) causes the loss of some of the proteins that are detected after washing with sodium carbonate, but it enables the detection of other hydrophobic proteins containing TMDs. Introduction of mass spectrometers with higher sensitivity, higher mass resolution and mass accuracy, and a faster scan rate significantly improved detection of membrane proteins, but the improved sample preparation is still useful and enables detection of additional hydrophobic proteins. Proteolytic predigestion of plasma membranes enables detection of additional hydrophobic proteins and better sequence coverage of TMD-containing proteins in plasma membranes from both tissues. [ABSTRACT FROM AUTHOR]

In proteomics, comprehensive analysis of peptides mixtures necessitates multiple dimensions of separation prior to mass spectrometry analysis to reduce sample complexity and increase the dynamic range of analysis. The main goal of this work was to improve the performance of (online) multidimensional protein identification technology (MudPIT) in terms of sensitivity, compatibility and recovery. The method employs weak anion and strong cation mixed-bed ion exchange chromatography (ACE) in the first separation dimension and reversed phase chromatography (RP) in the second separation dimension (Motoyama et.al. Anal. Chem 2007, 79, 3623-34.). We demonstrated that the chromatographic behavior of peptides in ACE chromatography depends on both the WAX/SCX mixing ratio as the ionic strength of the mobile phase system. This property allowed us to replace the conventional salt gradient by a (discontinuous) salt-free, pH gradient. First dimensional separation of peptides was accomplished with mixtures of aqueous formic acid and dimethylsulfoxide with increasing concentrations. The overall performance of this mobile phase system was found comparable to ammonium acetate buffers in application to ACE chromatography, but clearly outperformed strong cation exchange for use in first dimensional peptide separation. The dramatically improved compatibility between (salt-free) ion exchange chromatography and reversed phase chromatography-mass spectrometry allowed us to downscale the dimensions of the RP analytical column down to 25 μm i.d. for an additional 2- to 3-fold improvement in performance compared to current technology. The achieved levels of sensitivity, orthogonality, and compatibility demonstrates the potential of salt-free ACE MudPIT for the ultrasensitive, multidimensional analysis of very modest amounts of sample material. [ABSTRACT FROM AUTHOR]

Despite tremendous inroads in the development of more sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies for mass spectrometry-based proteomics, there remains a significant need for enhancing the selectivity of MS/MS-based workflows for streamlined analysis of complex biological mixtures. Here, a novel LC-MS/MS platform based on 351 nm ultraviolet photodissociation (UVPD) is presented for the selective analysis of cysteine-peptide subsets in complex protein digests. Cysteine-selective UVPD is mediated through the site-specific conjugation of reduced cysteine residues with a 351 nm active chromogenic Alexa Fluor 350 (AF350) maleimide tag. Only peptides containing the AF350 chromophore undergo photodissociation into extensive arrays of b- and y-type fragment ions, thus providing a facile means for differentiating cysteine-peptide targets from convoluting peptide backgrounds. With the use of this approach in addition to strategic proteolysis, the selective analysis of diagnostic heavy-chain complementarity determining regions (CDRs) of single-chain antibody (scAb) fragments is demonstrated. [ABSTRACT FROM AUTHOR]

In this study, we report the development of a microbore hollow fiber enzyme reactor (mHFER) coupled to nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) for the online digestion or selective enrichment of glycopeptides and analysis of proteins. With mHFER, enzymatic digestion of protein could be achieved by continuous flow within a very small volume (10 µL) of mHF inserted in a PEEK tube. Digested peptides exited through the pores of the hollow fiber membrane wall to external single or multiplexed trap columns for nLC-ESI-MS/MS analysis. Evaluation of online mHFER-nLC-ESI-MS/MS system was made with bovine serum albumin (BSA) by varying the temperature of digestion and the amount of protein injected. We evaluated the ability of the mHFER system to enrich glycopeptides by injecting a mixture of lectin (concanavalin A) and digested peptides from a-1-acid glycoprotein (AGP) into the mHFER, followed by delivery of PNGase F for endoglycosidic digestion. Nonglycosylated peptides unbound to lectins eluted at the first breakthrough run while N-linked glycopeptides eluted after the endoglycosidic digestion. The developed method was applied to urine samples from patients with prostate cancer and controls; 67 N-linked glycopeptides were identified and relative differences in glycopeptide content between patient and control samples were determined. [ABSTRACT FROM AUTHOR]

High field asymmetric waveform ion mobility spectrometry (FAIMS), also known as differential ion mobility spectrometry, coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) offers benefits for the analysis of complex proteomics samples. Advantages include increased dynamic range, increased signal-to-noise, and reduced interference from ions of similar m/ z. FAIMS also separates isomers and positional variants. An alternative, and more established, method of reducing sample complexity is prefractionation by use of strong cation exchange chromatography. Here, we have compared SCX-LC-MS/MS with LC-FAIMS-MS/MS for the identification of peptides and proteins from whole cell lysates from the breast carcinoma SUM52 cell line. Two FAIMS approaches are considered: (1) multiple compensation voltages within a single LC-MS/MS analysis (internal stepping) and (2) repeat LC-MS/MS analyses at different and fixed compensation voltages (external stepping). We also consider the consequence of the fragmentation method (electron transfer dissociation or collision-induced dissociation) on the workflow performance. The external stepping approach resulted in a greater number of protein and peptide identifications than the internal stepping approach for both ETD and CID MS/MS, suggesting that this should be the method of choice for FAIMS proteomics experiments. The overlap in protein identifications from the SCX method and the external FAIMS method was ~25 % for both ETD and CID, and for peptides was less than 20 %. The lack of overlap between FAIMS and SCX highlights the complementarity of the two techniques. Charge state analysis of the peptide assignments showed that the FAIMS approach identified a much greater proportion of triply-charged ions. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

High-throughput multiplexed proteomics of small- volume biospecimens will generate new opportunities in theranostics. Achieving parallel top-down and bottom-up mass spectrometry analyses of target proteins using a unified apparatus will improve proteome characterization. We have developed a novel silicon-based microfluidic device, multinozzle emitter array chip (MEA chip), as a new platform for small-volume proteomics using liquid chromatography-nanoelectrospray ionization mass spectrometry (LC-nanoESl- MS). We demonstrate parallel, on-chip, and online LC-MS analysis of hemoglobin and its tryptic digests directly from microliters of blood, achieving a detection limit of less than 5 red blood cells. Our MEA chip will enable clinical proteomics of small-volume samples. [ABSTRACT FROM AUTHOR]

Although two-dimensional electrophoresis (2D-GE) remains the basis for many ecotoxicoproteomic analyses, newer non-gel-based methods are beginning to be applied to overcome throughput and coverage limitations of 2D-GE. The overall objective of our research was to apply a comprehensive, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic approach to identity and quantify differentially expressed hepatic proteins from female fathead minnows exposed to fadrozole, a potent inhibitor of estrogen synthesis. Female fathead minnows were exposed to 0 (control), 0.04, and 1.0 μg of fadrozole/L of water for 4 days, and proteomic analysis was performed. Proteins were extracted and digested, and proteolytic peptides were separated via high-resolution one- or two-dimensional (1-D or 2-D) ultrapressure liquid chromatography (UPLC) and analyzed by tandem mass spectrometry. Mass spectra were searched against the National Center for Biotechnology Information (NCBI) ray-finned fish (Actinopterygii) database, resulting in identification of 782 unique proteins by single-dimension UPLC. When multidimensional LC analysis (2-D) was performed, an average increase of 1.9× in the number of identified proteins was observed. Differentially expressed proteins in fadrozole exposures were consistent with changes in liver function, including a decline in concentrations of vitellogenin as well as other proteins associated with endocrine function and cholesterol synthesis. Overall, these results demonstrate that a gel-free, label-free proteomic analysis method can successfully be utilized to determine differentially expressed proteins in small fish species after toxicant exposure. [ABSTRACT FROM AUTHOR]

Due to the hydrophilic nature of glycans, reverse phase chromatography has not been widely used as a glycomic separation technique coupled to mass spectrometry. Other approaches such as hydrophilic interaction chromatography and porous graphitized carbon chromatography are often employed, though these strategies frequently suffer from decreased chromatographic resolution, long equilibration times, indefinite retention, and column bleed. Herein, it is shown that, through an efficient hydrazone formation derivatization of N-linked glycans (4 h of additional sample preparation time which is carried out in parallel), numerous experimental and practical advantages are gained when analyzing the glycans by online reverse phase chromatography. These benefits include an increased number of glycans detected, increased peak capacity of the separation, and the ability to analyze glycans on the identical liquid chromatography-mass spectrometry platform commonly used for proteomic analyses. The data presented show that separation of derivatized N-linked glycans by reverse phase chromatography significantly out-performs traditional separation of native or derivatized glycans by hydrophilic interaction chromatography. Furthermore, the movement to a more ubiquitous separation technique will afford numerous research groups the opportunity to analyze both proteomic and glycomic samples on the same platform with minimal time and physical change between experiments, increasing the efficiency of "multiomic" biological approaches. [ABSTRACT FROM AUTHOR]

The functional design and application of a data-independent LC-MS precursor and product ion repository for protein identification, quantification, and validation is conceptually described. The ion repository was constructed from the sequence search results of a broad range of discovery experiments investigating various tissue types of two closely related mammalian species. The relative high degree of similarity in protein complement, ion detection, and peptide and protein identification allows for the analysis of normalized precursor and product ion intensity values, as well as standardized retention times, creating a multidimensional/orthogonal queryable, qualitative, and quantitative space. Peptide ion map selection for identification and quantification is primarily based on replication and limited variation. The information is stored in a relational database and is used to create peptide- and protein-specific fragment ion maps that can be queried in a targeted fashion against the raw or time aligned ion detections. These queries can be conducted either individually or as groups, where the latter affords pathway and molecular machinery analysis of the protein complement. The presented results also suggest that peptide ionization and fragmentation efficiencies are highly conserved between experiments and practically independent of the analyzed biological sample when using similar instrumentation. Moreover, the data illustrate only minor variation in ionization efficiency with amino acid sequence substitutions occurring between species. Finally, the data and the presented results illustrate how LC-MS performance metrics can be extracted and utilized to ensure optimal performance of the employed analytical workflows. [ABSTRACT FROM AUTHOR]

We report the effects of supercharging reagents dimethyl sulphoxide (DMSO) and m-nitrobenzyl alcohol ( m-NBA) applied to untargeted peptide identification, with special emphasis on non-tryptic peptides. Peptides generated from a mixture of five standard proteins digested with trypsin, elastase, or pepsin were separated with nanoflow liquid chromatography using mobile phases modified with either 5 % DMSO or 0.1 % m-NBA. Eluting peptides were ionized by online electrospray and sequenced by both CID and ETD using data-dependent MS/MS. Statistically significant improvements in peptide identifications were observed with DMSO co-solvent. In order to understand this observation, we assessed the effects of supercharging reagents on the chromatographic separation and the electrospray quality. The increase in identifications was not due to supercharging, which was greater for the 0.1 % m-NBA co-solvent and not observed for the 5.0 % DMSO co-solvent. The improved MS/MS efficiency using the DMSO modified mobile phase appeared to result from charge state coalescence. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The use of narrow bore LC capillaries operated at altralow flow rates coupled with mass spectrometay provides a desirable convergence of figures of merit to support high- performance LC-MS/MS analysis. This configuration provides a viable means to achieve in-depth protein sequence coverage while maintaining a high rate of data production. Here we explore potential performance improvements afforded by use of 25 μm x 100 cm columns fabricated with 5 μdiameter reversed phase particles and integrated electrospray emitter tips. These columns achieve a separation peak capacity of ≈750 m a 600-mm gradient, with average chromatographic peak widths of less than 1 cain. At room temperature, a pressure drop of only es1500 psi is sufficient to maintain an effluent flow rate of ⩽10 nL/min. Using mouse embryonic stem cells as a model for complex mammalian proteomes, we reproducibly identify over 4000 proteins across duplicate 600 mm LC-MS/MS analyses. [ABSTRACT FROM AUTHOR]

Sodium dodecyl sulfate (SDS) is one of the most popular laboratory reagents used for biological sample extraction; however, the presence of this reagent in samples challenges LC-MS-based proteomics analyses because it can interfere with reversed-phase LC separations and electrospray ionization. This study reports a simple SDS-assisted proteomics sample preparation method facilitated by a novel peptide-level SDS removal step. In an initial demonstration, SDS was effectively (>99.9%) removed from peptide samples through ion substitution-mediated DS- precipitation using potassium chloride (KCl), and excellent peptide recovery (>95%) was observed for <20 μg of peptides. Further experiments demonstrated the compatibility of this protocol with LC-MS/MS analyses. The resulting proteome coverage obtained for both mammalian tissues and bacterial samples was comparable to or better than that obtained for the same sample types prepared using standard proteomics preparation methods and analyzed using LC-MS/MS. These results suggest the SDS-assisted protocol is a practical, simple, and broadly applicable proteomics sample processing method, which can be particularly useful when dealing with samples difficult to solubilize by other methods. [ABSTRACT FROM AUTHOR]

Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ∼70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa. [ABSTRACT FROM AUTHOR]

We demonstrate the use of capillary zone electrophoresis with an electrokinetically pumped sheath-flow electrospray interface for the analysis of a tryptic digest of a sample of intermediate protein complexity, the secreted protein fraction of Mycobacterium marinum. For electrophoretic analysis, 11 fractions were generated from the sample using reverse-phase liquid chromatography; each fraction was analyzed by CZE-ESI-MS/MS, and 334 peptides corresponding to 140 proteins were identified in 165 min of mass spectrometer time at 95% confidence (FDR < 0.15%). In comparison, 388 peptides corresponding to 134 proteins were identified in 180 min of mass spectrometer time by triplicate UPLC-ESI-MS/MS analyses, each using 250 ng of the unfractionated peptide mixture, at 95% confidence (FDR < 0.15%). Overall, 62% of peptides identified in CZE-ESI-MS/MS and 67% in UPLC-ESI-MS/MS were unique. CZE-ESI-MS/MS favored basic and hydrophilic peptides with low molecular masses. Combining the two data sets increased the number of unique peptides by 53%. Our approach identified more than twice as many proteins as the previous record for capillary electrophoresis proteome analysis. CE-ESI-MS/MS is a useful tool for the analysis of proteome samples of intermediate complexity. [ABSTRACT FROM AUTHOR]