Rapid direct detection of the major fish allergen, parvalbumin, by selected MS/MS ion monitoring mass spectrometry

Parvalbumins beta (β-PRVBs) are considered the major fish allergens. A new strategy for the rapid and direct detection of these allergens in any foodstuff is presented in this work. The proposed methodology is based on the purification of β-PRVBs by treatment with heat, the use of accelerated in-solution trypsin digestion under an ultrasonic field provided by High-Intensity Focused Ultrasound (HIFU) and the monitoring of only nineteen β-PRVB peptide biomarkers by Selected MS/MS Ion Monitoring (SMIM) in a linear ion trap (LIT) mass spectrometer. The present strategy allows the direct detection of the presence of fish β-PRVBs in any food product in less than 2 hours.     

Evaluation of blood collection tubes using selected reaction monitoring MS: Implications for proteomic biomarker studies

An emphasis of current proteomic research is the validation of plasma protein biomarkers. The process of blood collection itself is critical to the accuracy and reproducibility of quantitative biomarker assays. We have developed selected reaction monitoring (SRM) assays to analyse thirteen abundant plasma proteins and evaluated the impact of three different blood collection tubes on the levels of these proteins. We also assessed the implications of the time taken to analyse plasma samples by evaluating the recovery of these proteins. We showed that SRM detects minor differences in the levels of some proteins which can be attributed to collection tube type. The average recovery for 12 of 18 assays was higher for proteins that were collected in tubes containing protease inhibitors compared to conventional collection tubes. For five of the assays, the differential recovery was statistically significant. Delaying MS analysis of a freeze‐thawed sample for 1 hour showed greatly reduced recovery of these analytes; however differences attributed to tube type were only evident at the baseline timepoint. Finally, we assessed the natural variation of circulating levels of these proteins in a cohort of seven healthy individuals. This study provides useful information for researchers contemplating blood collection for undertaking protein biomarker studies.     

A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry

The majority of eukaryotic proteins are phosphorylated in vivo, and phosphorylation may be the most common regulatory posttranslational modification. Many proteins are phosphorylated at numerous sites, often by multiple kinases, which may have different functional consequences. Understanding biological functions of phosphorylation events requires methods to detect and quantify individual sites within a substrate. Here we outline a general strategy that addresses this need and relies on the high sensitivity and specificity of selected reaction monitoring (SRM) mass spectrometry, making it potentially useful for studying in vivo phosphorylation without the need to isolate target proteins. Our approach uses label-free quantification for simplicity and general applicability, although it is equally compatible with stable isotope quantification methods. We demonstrate that label-free SRM-based quantification is comparable to conventional assays for measuring the kinetics of phosphatase and kinase reactions in vitro. We also demonstrate the capability of this method to simultaneously measure relative rates of phosphorylation and dephosphorylation of substrate mixtures, including individual sites on intact protein substrates in the context of a whole cell extract. This strategy should be particularly useful for characterizing the physiological substrate specificity of kinases and phosphatases and can be applied to studies of other protein modifications as well.     

Range of protein detection by selected/multiple reaction monitoring mass spectrometry in an unfractionated human cell culture lysate

Selected or multiple reaction monitoring is a targeted mass spectrometry method (S/MRM-MS), in which many peptides are simultaneously and consistently analyzed during a single liquid chromatography-mass spectrometry (LC-S/MRM-MS) measurement. These capabilities make S/MRM-MS an attractive method to monitor a consistent set of proteins over various experimental conditions. To increase throughput for S/MRM-MS it is advantageous to use scheduled methods and unfractionated protein extracts. Here, we established the practically measurable dynamic range of proteins reliably detectable and quantifiable in an unfractionated protein extract from a human cell line using LC-S/MRM-MS. Initially, we analyzed S/MRM transition peak groups in terms of interfering signals and compared S/MRM transition peak groups to MS1-triggered MS2 spectra using dot-product analysis. Finally, using unfractionated protein extract from human cell lysate, we quantified the upper boundary of copies per cell to be 35 million copies per cell, while 7500 copies per cell represents a lower boundary using a single 35 min linear gradient LC-S/MRM-MS measurement on a current, standard commercial instrument.     

Review of application of mass spectrometry for analyses of anterior eye proteome

Proteins have important functional roles in the body, which can be altered in disease states. The eye is a complex organ rich in proteins; in particular, the anterior eye is very sophisticated in function and is most commonly involved in ophthalmic diseases. Proteomics, the large scale study of proteins, has greatly impacted our knowledge and understanding of gene function in the post-genomic period. The most significant breakthrough in proteomics has been mass spectrometric identification of proteins, which extends analysis far beyond the mere display of proteins that classical techniques provide. Mass spectrometry functions as a "mass analyzer" which simplifies the identification and quantification of proteins extracted from biological tissue. Mass spectrometric analysis of the anterior eye proteome provides a differential display for protein comparison of normal and diseased tissue. In this article wepresent the key proteomic findings in the recent literature related to the cornea, aqueous humor, trabecular meshwork, iris, ciliary body and lens. Through this we identified unique proteins specific to diseases related to the anterior eye.     

The development of selected reaction monitoring methods for targeted proteomics via empirical refinement

Software advancements in the last several years have had a significant impact on proteomics from method development to data analysis. Herein, we detail a method, which uses our in-house developed software tool termed Skyline, for empirical refinement of candidate peptides from targeted proteins. The method consists of four main steps from generation of a testable hypothesis, method development, peptide refinement, to peptide validation. The ultimate goal is to identify the best performing peptide in terms of ionization efficiency, reproducibility, specificity, and chromatographic characteristics to monitor as a proxy for protein abundance. It is important to emphasize that this method allows the user to perform this refinement procedure in the sample matrix and organism of interest with the instrumentation available. Finally, the method is demonstrated in a case study to determine the best peptide to monitor the abundance of surfactant protein B in lung aspirates.     

Selectivity of bacterial proteome fractionation based on differential solubility: a mass spectrometry evaluation

We investigate the selectivity achieved after differential solubilization of bacterial proteomes following two procedures, both based on successive extraction of proteins in solutions of increasing solubilizing power. Recently, these procedures have gained notable popularity and several commercial kits are now available. A total of 225 proteins in one case and 227 proteins in the other were identified by LC MSMS analysis; 146 of them were identified in both procedures. The proportions of proteins identified as present in only one fraction were 64 and 57%, respectively. The distribution of cytosolic, membrane, and ribosomal proteins among the successive extracts was analyzed in detail. The effect of (1) replacement of low-speed with high-speed centrifugation, (2) omission of detergents in urea solutions, (3) successive washes of pellets, and (4) reproducibility was evaluated. Proteins with positive grand averages of hydropathicity values and membrane proteins were found in all fractions. This study highlights the benefits and limitations of differential solubilization methods, focusing on practical aspects that may strongly influence their selectivity.     

An assessment of current bioinformatic solutions for analyzing LC-MS data acquired by selected reaction monitoring technology

Selected reaction monitoring (SRM) is an accurate quantitative technique, typically used for small-molecule mass spectrometry (MS). SRM has emerged as an important technique for targeted and hypothesis-driven proteomic research, and is becoming the reference method for protein quantification in complex biological samples. SRM offers high selectivity, a lower limit of detection and improved reproducibility, compared to conventional shot-gun-based tandem MS (LC-MS/MS) methods. Unlike LC-MS/MS, which requires computationally intensive informatic postanalysis, SRM requires preacquisition bioinformatic analysis to determine proteotypic peptides and optimal transitions to uniquely identify and to accurately quantitate proteins of interest. Extensive arrays of bioinformatics software tools, both web-based and stand-alone, have been published to assist researchers to determine optimal peptides and transition sets. The transitions are oftentimes selected based on preferred precursor charge state, peptide molecular weight, hydrophobicity, fragmentation pattern at a given collision energy (CE), and instrumentation chosen. Validation of the selected transitions for each peptide is critical since peptide performance varies depending on the mass spectrometer used. In this review, we provide an overview of open source and commercial bioinformatic tools for analyzing LC-MS data acquired by SRM.     

The rat brain hippocampus proteome

The hippocampus is crucial in memory storage and retrieval and plays an important role in stress response. In humans, the CA1 area of hippocampus is one of the first brain areas to display pathology in Alzheimer's disease. A comprehensive analysis of the hippocampus proteome has not been accomplished yet. We applied proteomics technologies to construct a two-dimensional database for rat brain hippocampus proteins. Hippocampus samples from eight months old animals were analyzed by two-dimensional electrophoresis and the proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The database comprises 148 different gene products, which are in the majority enzymes, structural proteins and heat shock proteins. It also includes 39 neuron specific gene products. The database may be useful in animal model studies of neurological disorders.     

The application of robotics and mass spectrometry to the characterisation of the Drosophila melanogaster indirect flight muscle proteome

The rapid accumulation of sequence data generated by the various genome sequencingprojects and the generation of expressed sequence tag databases has resulted in the need forthe development of fast and sensitive methods for the identification and characterisation oflarge numbers of gel electrophoretically separated proteins to translate the sequence data intobiological function. To achieve this goal it has been necessary to devise new approaches toprotein analysis: matrix-assisted laser desorption and electrospray mass spectrometry havebecome important protein analytical tools which are both fast and sensitive. When combinedwith a robotic system for the in-gel digestion of electrophoretically separated proteins, itbecomes possible to rapidly identify many proteins by searching databases with MS data. Thepower of this combination of techniques is demonstrated by an analysis of the proteins presentin the myofibrillar lattice of the indirect flight muscle of Drosophila melanogaster. Theproteins were separated by SDS-PAGE and in-gel proteolysis was performed bothautomatically and manually. All 16 major proteins could quickly be identified by massspectrometry. Although most of the protein components were known to be present in theflight muscle, two new components were also identified. The combination of methodsdescribed offers a means for the rapid identification of large numbers of gel separatedproteins.     

Identification of psoriatic arthritis mediators in synovial fluid by quantitative mass spectrometry

Background

Synovial fluid (SF) is a dynamic reservoir for proteins originating from the synovial membrane, cartilage, and plasma, and may therefore reflect the pathophysiological conditions that give rise to arthritis. Our goal was to identify and quantify protein mediators of psoriatic arthritis (PsA) in SF.

Methods

Age and gender-matched pooled SF samples from 10 PsA and 10 controls [early osteoarthritis (OA)], were subjected to label-free quantitative proteomics using liquid chromatography coupled to mass spectrometry (LC-MS/MS), to identify differentially expressed proteins based on the ratios of the extracted ion current of each protein between the two groups. Pathway analysis and public database searches were conducted to ensure these proteins held relevance to PsA. Multiplexed selected reaction monitoring (SRM) assays were then utilized to confirm the elevated proteins in the discovery samples and in an independent set of samples from patients with PsA and controls.

Results

We determined that 137 proteins were differentially expressed between PsA and control SF, and 44 were upregulated. The pathways associated with these proteins were acute-phase response signalling, granulocyte adhesion and diapedesis, and production of nitric oxide and reactive oxygen species in macrophages. The expression of 12 proteins was subsequently quantified using SRM assays.

Conclusions

Our in-depth proteomic analysis of the PSA SF proteome identified 12 proteins which were significantly elevated in PsA SF compared to early OA SF. These proteins may be linked to the pathogenesis of PsA, as well serve as putative biomarkers and/or therapeutic targets for this disease.     

Quantifying proteins by mass spectrometry: The selectivity of SRM is only part of the problem

Precise and accurate protein quantification is critical to many areas of proteomics. Antibody‐based approaches are costly and time‐consuming to develop, consequently, there is considerable interest in alternative quantitative methods that are versatile and can be implemented without the considerable delays associated with antibody development and characterization. Approaches based on MS have therefore attracted considerable attention and are now frequently touted as the most practical and powerful of all options. Nevertheless, there are serious limitations associated with quantifying a protein based on tandem mass analysis of one or two peptides generated by either chemical or enzymatic cleavage. In an accompanying Viewpoint article, Molloy and coworkers point out that selectivity is not necessarily guaranteed despite the power of SRM. Here we address an additional concern that can also compromise specificity. In complex mammalian systems, multiple proteins can serve as precursors of a single peptide and consequently, depending on the peptide(s) selected, protein levels may be significantly under‐ or overestimated.     

Miniaturized proteomics and peptidomics using capillary liquid separation and high resolution mass spectrometry

Knowledge of the protein and peptide content in a tissue or a body fluid is vital in many areas of medical and biomedical sciences. Information from proteomic and peptidomic studies may reveal alterations in expression due to, e.g., a disease and facilitate the understanding of the pathophysiology and the identification of biological markers. In this minireview, we discuss miniaturized proteomic and peptidomic approaches that have been applied in our laboratory in order to investigate the protein and peptide contents of body fluids (such as plasma, cerebrospinal and amniotic fluid), as well as extracted tissues. The methods involve miniaturized liquid separation, i.e., capillary liquid chromatography and capillary electrophoresis, combined with high resolution mass spectrometry (MS), i.e., Fourier transform ion cyclotron resonance MS. These approaches provide the opportunity to analyze samples of small volumes with high throughput, high sensitivity, good dynamic range and minimal sample handling. Also, the experiments are relatively easy to automate.     

Near infrared spectroscopy compared to liquid chromatography coupled to mass spectrometry and capillary electrophoresis as a detection tool for peptide reaction monitoring

Peptide interaction is normally monitored by liquid chromatography (LC), liquid chromatography coupled to mass spectrometry (LC-MS), mass spectrometric (MS) methods such as MALDI-TOF/MS or capillary electrophoresis (CE). These analytical techniques need to apply either high pressure or high voltages, which can cause cleavage of newly formed bondages. Therefore, near infrared reflectance spectroscopy (NIRS) is presented as a rapid alternative to monitor the interaction of glutathione and oxytocin, simulating physiological conditions. Thereby, glutathione can act as a nucleophile with oxytocin forming four new conjugates via a disulphide bondage. Liquid chromatography coupled to UV (LC-UV) and mass spectrometry via an electrospray ionisation interface (LC-ESI-MS) resulted in a 82% and a 78% degradation of oxytocin at pH 3 and a 5% and a 7% degradation at pH 6.5. Capillary electrophoresis employing UV-detection (CE-UV) showed a 44% degradation of oxytocin. LC and CE in addition to the NIRS are found to be authentic tools for quantitative analysis. Nevertheless, NIRS proved to be highly suitable for the detection of newly formed conjugates after separating them on a thin layer chromatography (TLC) plate. The recorded fingerprint in the near infrared region allows for a selective distinct qualitative identification of conjugates without the need for expensive instrumentation such as quadrupole or MALDI-TOF mass spectrometers. The performance of the established NIRS method is compared to LC and CE; its advantages are discussed in detail.     

High-throughput characterization of lipopolysaccharide-binding proteins using mass spectrometry

Lipopolysaccharide (LPS)-binding proteins interact with LPS in human serum and mediate various immune responses. We describe a high-throughput LPS-binding protein profiling platform for discovering unknown LPS-binding proteins and potential inflammatory mediators. As a pull-down method, the LPS molecules were immobilized onto epoxy beads and then directly incubated with human serum to screen LPS-binding proteins. Through the "untargeted" mass spectrometric approach, potential LPS-binding proteins which elicit various immune responses in human serum were identified by a highly sensitive LTQ Orbitrap Hybrid Fourier Transform Mass Spectrometer (LTQ Orbitrap FT MS). Therefore, this mass spectrometry (MS)-based profiling method is straightforward for screening unknown LPS-binding proteins and provides physiologically relevant binding partners in human serum.     

Use of selected reaction monitoring mass spectrometry for the detection of specific MHC class I peptide antigens on A3 supertype family members

The development of peptide-based vaccines that are useful in the therapeutic treatment of melanoma and other cancers ultimately requires the identification of a sufficient number of antigenic peptides so that most individuals, regardless of their major histocompatibility complex (MHC)–encoded class I molecule phenotype, can develop a cytotoxic T lymphocyte (CTL) response against one or more peptide components of the vaccine. While it is relatively easy to identify antigenic peptides that are presented by the most prevalent MHC class I molecules in the population, it is problematic to identify antigenic peptides that are presented by MHC class I molecules that have less frequent expression in the population. One manner in which this problem can be overcome is by taking advantage of known MHC class I supertypes, which are groupings of MHC class I molecules that bind peptides sharing a common motif. We have developed a mass spectrometric approach which can be used to determine if an antigenic peptide is naturally processed and presented by any given MHC class I molecule. This approach has been applied to the A3 supertype, and the results demonstrate that some, but not all, A3 supertype family–associated peptides can associate with all A3 supertype family members. The approach also demonstrates the shared nature of several newly identified peptide antigens. The use of this technology negates the need to test peptides for their ability to stimulate CTL responses in those cases where the peptide is not naturally processed and bound to the target MHC class I molecule of interest, thus allowing resources to be focused on the most promising vaccine candidates.     

A preliminary investigation of the Nostoc punctiforme proteome

Nostoc punctiforme ATCC 29133 is a filamentous terrestrial cyanobacterium (prokaryote) that expresses several different phenotypes in response to environmental cues. When grown in nitrogen-deficient media the most abundant proteins in addition to phycobiliproteins were superoxide dismutase, ATP synthase, and peptidyl-prolyl cis-trans isomerases. A methylated peptide from an akinete marker protein was also identified, suggesting that methylation could potentially play a regulatory role through signaling. C-phycocyanin alpha-chain was methylated at the C-terminal end of the protein and tandem mass spectrometric data also identified peptides that were deamidated. Since a significant number of putative polyketide/non-ribosomal peptide synthase genes are present in the annotated genome, an analysis of a methanolic extract of whole cells was also performed, and a series of nostopeptolides were identified.     

Targeted proteomics by selected reaction monitoring mass spectrometry: applications to systems biology and biomarker discovery

Mass Spectrometry-based proteomics is now considered a relatively established strategy for protein analysis, ranging from global expression profiling to the identification of protein complexes and specific post-translational modifications. Recently, Selected Reaction Monitoring Mass Spectrometry (SRM-MS) has become increasingly popular in proteome research for the targeted quantification of proteins and post-translational modifications. Using triple quadrupole instrumentation (QqQ), specific analyte molecules are targeted in a data-directed mode. Used routinely for the quantitative analysis of small molecular compounds for at least three decades, the technology is now experiencing broadened application in the proteomics community. In the current review, we will provide a detailed summary of current developments in targeted proteomics, including some of the recent applications to biological research and biomarker discovery.