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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Gel absorption-based sample preparation for the analysis of membrane proteome by mass spectrometry

A gel absorption-based sample preparation method for shotgun analysis of membrane proteome has been developed. In this new method, membrane proteins solubilized in a starting buffer containing a high concentration of sodium dodecyl sulfate (SDS) were directly entrapped and immobilized into gel matrix when the membrane protein solution was absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts were removed by washing, the proteins were subjected to in-gel digestion and the tryptic peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). The results showed that the newly developed method not only avoided the protein loss and the adverse protein modifications during gel embedment but also improved the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides, thereby facilitating the identification of membrane proteins, especially the integral membrane proteins. Compared with the conventional tube-gel digestion method, the newly developed method increased the numbers of identified membrane proteins and integral membrane proteins by 25.0% and 30.2%, respectively, demonstrating that the method is of broad practicability in gel-based shotgun analysis of membrane proteome.     

Analysis of the Listeria cell wall proteome by two-dimensional nanoliquid chromatography coupled to mass spectrometry

Genome analyses have revealed that the Gram-positive bacterial species Listeria monocytogenes and L. innocua contain a large number of genes encoding surface proteins predicted to be covalently bound to the cell wall (41 and 34, respectively). The function of most of these proteins is unknown and they have not even been identified biochemically. Here, we report the first characterization of the Listeria cell wall proteome using a nonelectrophoretic approach. The material analyzed consisted of a peptide mixture obtained from a cell wall extract insoluble in boiling 4% SDS. This extract, containing peptidoglycan (intrinsically resistant to proteases) and strongly associated proteins, was digested with trypsin in a solution with 0.01% SDS, used to favor protein digestion throughout the peptidoglycan. The resulting complex peptide mixture was fractionated and analyzed by two-dimensional nanoliquid chromatography coupled to ion-trap mass spectrometry. A total of 30 protein species were unequivocally identified in cell wall extracts of the genome strains L. monocytogenes EGD-e (19 proteins) and L. innocua CLIP11262 (11 proteins). Among them, 20 proteins bearing an LPXTG motif recognized for covalent anchoring to the peptidoglycan were identified. Other proteins detected included peptidoglycan-lytic enzymes, a penicillin-binding protein, and proteins bearing an NXZTN motif recently proposed to direct protein anchoring to the peptidoglycan. The marked sensitivity of the method makes it highly attractive in the post-genome era for defining the cell wall proteome in any bacterial species. This information will be useful to study novel protein-peptidoglycan associations and to rapidly identify new targets in the surface of important bacterial pathogens.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry protocol for monitoring the progress of enzymatic (13)C/(15)N-labeled DNA syntheses

We demonstrate that a simple matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry protocol provides a rapid and accurate method for monitoring the stage and completeness of enzymatic DNA syntheses. A crucial step of these syntheses is to quench the reaction at the desired nucleotide length. This is especially important when expensive, e.g., (13)C/(15)N-labeled DNA segments, are synthesized for multinuclear magnetic resonance purposes to reveal detailed structural information. The analyses of three templates for a human telomeric 22-mer, a wild type, and a mutant human c-MYC promoter (18- and 22-mer) DNA and their reactions with the 3'-5' exo(-) Klenow fragment of DNA polymerase I demonstrate the usefulness of our protocol. Small amounts of samples (ca. 1-2 microl each) were taken from the reaction mixtures at different times and analyzed promptly by MALDI-TOF, applying our successive on-plate desalting method that eliminates the insensitivity of the MALDI technique at high salt content. The progress of the reaction was detected by monitoring the relative intensity ratios of ions corresponding to the desired products and the primer-template complexes. The effectiveness of NH(3) cleavage leading to final products was also followed by MALDI-TOF in successful enzymatic reactions.     

Synthesis of d-labeled and unlabeled benzoyloxysuccinimides and application to quantitative analysis of peptides and a protein by isotope differential mass spectrometry

Benzoyloxysuccinimide and its d₅-labeled version, which react with amino groups in the N-termini and lysine side chains in proteins, were synthesized and applied to quantitative analysis of peptides and a commercially available protein in combination with a MALDI mass spectrometer.     

Thematic minireview series on biological applications of mass spectrometry

Mass spectrometry is a powerful technique with many applications in biology as well as chemistry and physics. The increases in sensitivity and resolution of the instruments, coupled with improvements in the analysis of data, have opened new dimensions in analyses of complex biological systems. Examples presented here include drug metabolism, lipid analysis, metabolomics, quantitative proteomics, direct analysis of intact proteins, and imaging of both small molecules and proteins in tissues.     

In situ metabolomic mass spectrometry imaging: recent advances and difficulties

MS imaging (MSI) is a remarkable new technology that enables us to determine the distribution of biological molecules present in tissue sections by direct ionization and detection. This technique is now widely used for in situ imaging of endogenous or exogenous molecules such as proteins, lipids, drugs and their metabolites, and it is a potential tool for pathological analysis and the investigation of disease mechanisms. MSI is also thought to be a technique that could be used for biomarker discovery with spatial information. The application of MSI to the study of endogenous metabolites has received considerable attention because metabolites are the result of the interactions of a system's genome with its environment and a total set of these metabolites more closely represents the phenotype of an organism under a given set of conditions. Recent studies have suggested the importance of in situ metabolite imaging in biological discovery and biomedical applications, but several issues regarding the technical application limits of MSI still remained to be resolved. In this review, we describe the capabilities of the latest MSI techniques for the imaging of endogenous metabolites in biological samples, and also discuss the technical problems and new challenges that need to be addressed for effective and widespread application of MSI in both preclinical and clinical settings.     

Reproducibility of mass spectrometry based protein profiles for diagnosis of ovarian cancer across clinical studies: A systematic review

The focus of this systematic review is to give an overview of the current status of clinical protein profiling studies using MALDI and SELDI MS platforms in the search for ovarian cancer biomarkers. A total of 34 profiling studies were qualified for inclusion in the review. Comparative analysis of published discriminatory peaks to peaks found in an original MALDI MS protein profiling study was made to address the key question of reproducibility across studies. An overlap was found despite substantial heterogeneity between studies relating to study design, biological material, pre-analytical treatment, and data analysis. About 47% of the peaks reported to be associated to ovarian cancer were also represented in our experimental study, and 34% of these redetected peaks also showed a significant difference between cases and controls in our study. Thus, despite known problems related to reproducibility an overlap in peaks between clinical studies was demonstrated, which indicate convergence toward a set of common discriminating, reproducible peaks for ovarian cancer. The potential of the discriminating protein peaks for clinical use as ovarian cancer biomarkers will be discussed and evaluated. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Linear and accurate quantitation of proenkephalin-derived peptides by isotopic labeling with internal standards and mass spectrometry

Proenkephalin (PE) represents the precursor protein of the active peptide neurotransmitter enkephalin. Quantitative analysis of peptides and proteins is an objective of mass spectrometry-based studies of biological systems and will be important for studying the proteolytic conversion of proproteins to active enkephalin and neuropeptides. The goal of this study was to define and optimize quantitation of different amounts of tryptic peptides derived from PE using light (H4, 4 hydrogens) and heavy (D4, 4 deuteriums) succinic anhydride for isotopic labeling of peptides analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Comparisons were made between PE-derived peptides with and without internal standards. Importantly, incorporation of internal standards of known amounts of heavy isotope-labeled tryptic peptides of PE provided linear calibration plots with accurate quantitation. In contrast, comparison of light and heavy isotope-labeled peptides without internal standards produced variable and inaccurate nonlinear isotopic ratio comparisons of PE-derived peptides. These results demonstrate that use of internal standards composed of a defined amount(s) of standard peptides (PE-derived tryptic peptides) is necessary for high-quality linear quantitation of peptides by isotopic labeling and MS/MS.