Interpretation of mass spectrometry data for high-throughput proteomics

Recent developments in proteomics have revealed a bottleneck in bioinformatics: high-quality interpretation of acquired MS data. The ability to generate thousands of MS spectra per day, and the demand for this, makes manual methods inadequate for analysis and underlines the need to transfer the advanced capabilities of an expert human user into sophisticated MS interpretation algorithms. The identification rate in current high-throughput proteomics studies is not only a matter of instrumentation. We present software for high-throughput PMF identification, which enables robust and confident protein identification at higher rates. This has been achieved by automated calibration, peak rejection, and use of a meta search approach which employs various PMF search engines. The automatic calibration consists of a dynamic, spectral information-dependent algorithm, which combines various known calibration methods and iteratively establishes an optimised calibration. The peak rejection algorithm filters signals that are unrelated to the analysed protein by use of automatically generated and dataset-dependent exclusion lists. In the "meta search" several known PMF search engines are triggered and their results are merged by use of a meta score. The significance of the meta score was assessed by simulation of PMF identification with 10,000 artificial spectra resembling a data situation close to the measured dataset. By means of this simulation the meta score is linked to expectation values as a statistical measure. The presented software is part of the proteome database ProteinScape which links the information derived from MS data to other relevant proteomics data. We demonstrate the performance of the presented system with MS data from 1891 PMF spectra. As a result of automatic calibration and peak rejection the identification rate increased from 6% to 44%.Abbreviations 2-DE Two-dimensional gel electrophoresis - MALDI Matrix-assisted laser desorption ionisation - PMF Peptide mass fingerprinting - MS Mass spectrometry - TOF Time of flight     

A mass spectrometry approach for the study of deglycosylated proteins

Mass spectrometry (MS) analysis, after enzymatic or chemical deglycosylation, requires preparatory steps to remove salts and buffers. In this work, the glycosylated protein fetuin and a lectin protein isolated from the serum of Alligator mississippiensis were used to evaluate methods for desalting samples after an enzymatic or chemical deglycosylation. Precipitation and dialysis were used to prepare the deglycosylated samples for MS analysis. Both the precipitation and dialysis methods were suitable for sample preparation prior to analysis by matrix assisted laser desorption ionization (MALDI) MS.     

Low-molecular weight protein profiling of genetically modified maize using fast liquid chromatography electrospray ionization and time-of-flight mass spectrometry

In this work, the use of liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC-TOFMS) has been evaluated for the profiling of relatively low-molecular weight protein species in both genetically modified (GM) and non-GM maize. The proposed approach consisted of a straightforward sample fractionation with different water and ethanol-based buffer solutions followed by separation and detection of the protein species using liquid chromatography with a small particle size (1.8 μm) C(18) column and electrospray-time-of-flight mass spectrometry detection in the positive ionization mode. The fractionation of maize reference material containing different content of transgenic material (from 0 to 5% GM) led to five different fractions (albumins, globulins, zeins, zein-like glutelins, and glutelins), all of them containing different protein species (from 2 to 52 different species in each fraction). Some relevant differences in the quantity and types of protein species were observed in the different fractions of the reference material (with different GM contents) tested, thus revealing the potential use of the proposed approach for fast protein profiling and to detect tentative GMO markers in maize.     

The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites

Protein phosphorylation analysis is an enormous challenge. This review summarises the currently used techniques, which are based on radiolabelling and mass spectrometry as well as electrophoretic and chromatographic separation. Many methods exist, but there is still no single procedure applicable to all phosphoproteins. MS is able to deliver information about the location of phosphorylation sites, but phosphospecific properties with respect to ionisation present obstacles. Therefore, multidimensional approaches involving several analytical methods are often necessary to conquer phosphorylation site identification.Abbreviations 2D Two-dimensional - CE Capillary electrophoresis - CID Collision-induced dissociation - ECD Electron capture dissociation - ESI Electrospray ionisation - FT-ICR Fourier transform ion cyclotron resonance - HPLC High performance liquid chromatography - ICAT Isotope coded affinity tags - ICP Inductively-coupled plasma - IDA Immino-diacetic acid - IMAC Immobilised metal affinity chromatography - IRMPD Infrared multiphoton dissociation - IT Ion trap - MALDI Matrix-assisted laser desorption/ionisation - MRP14 Myeloid-related protein 14 - MS Mass spectrometry - NTA Nitrilo-triacetic acid - PAGE Polyacrylamide gel electrophoresis - PDI Protein disulfide isomerase - pS Phosphoserine residue - PSD Post-source decay - pT Phosphothreonine residue - PVDF Polyvinylidene fluoride - pY Phosphotyrosine residue - Q-TOF Quadrupole-time-of-flight - RP Reversed phase - SIM Single-ion monitoring - SDS Sodium dodecyl sulfate - SORI Sustained off-resonance irradiation - TLC Thin-layer chromatography - TOF Time-of-flight An erratum to this article can be found at     

Verification of protein biomarker specificity for the identification of biological stains by quadrupole time‐of‐flight mass spectrometry

Advances in proteomics technology over the past decade offer forensic serologists a greatly improved opportunity to accurately characterize the tissue source from which a DNA profile has been developed. Such information can provide critical context to evidence and can help to prioritize downstream DNA analyses. Previous proteome studies compiled panels of “candidate biomarkers” specific to each of five body fluids (i.e ., peripheral blood, vaginal/menstrual fluid, seminal fluid, urine, and saliva). Here, a multiplex quadrupole time‐of‐flight mass spectrometry assay has been developed in order to verify the tissue/body fluid specificity the 23 protein biomarkers that comprise these panels and the consistency with which they can be detected across a sample population of 50 humans. Single‐source samples of these human body fluids were accurately identified by the detection of one or more high‐specificity biomarkers. Recovery of body fluid samples from a variety of substrates did not impede accurate characterization and, of the potential inhibitors assayed, only chewing tobacco juice appeared to preclude the identification of a target body fluid. Using a series of 2‐component mixtures of human body fluids, the multiplex assay accurately identified both components in a single‐pass. Only in the case of saliva and peripheral blood did matrix effects appear to impede the detection of salivary proteins.     

Fast on-column protein digestion with subsequent peptide mapping using tandem mass spectrometry with information dependent acquisition

A platform for rapid on-line protein digestion of protein mixtures for direct infusion to a mass spectrometer is presented. A mixture of protein A, staphylococcal enterotoxin B and cytochrome c was used as a model mixture injected on a gel filtration column and a trypsin reactor which were connected in series to a micro liquid chromatography (microLC) system. The peptides in the column eluate were analyzed with ESI tandem mass spectrometry, utilizing information dependent acquisition (IDA). In one step, the proteins in the mixture (microM concentrations) were concomitantly desalted, separated, digested and identified with an overall analysis time of less than 40 min. Protein sequence coverage of 78-95% for the involved substances was achieved.     

Elemental mass spectrometry for quantitative proteomics

In the last decade mass-spectrometry-based proteomics has become an indispensable analytical tool for molecular biology, cellular biology and, lately, for the emerging systems biology. This review summarises the evolution and great potential of analytical methods based on elemental mass-spectrometric detection for quantitative proteomic analysis.     

Probing protein dynamics and function under native and mildly denaturing conditions with hydrogen exchange and mass spectrometry

A combination of hydrogen exchange and mass spectrometry emerged in recent years as a powerful experimental tool capable of probing both structural and dynamic features of proteins. Although its concept is very simple, the interpretation of experimental data is not always straightforward, as a combination of chemical reactions (isotope exchange) and dynamic processes within protein molecules give rise to convoluted exchange patterns. This paper provides a historical background of this technique, candid assessment of its current state and limitations and a discussion of promising recent developments that can result in tremendous improvements and a dramatic expansion of the scope of its applications.     

Analysis of food proteins and peptides by mass spectrometry-based techniques

Mass spectrometry has arguably become the core technology for the characterization of food proteins and peptides. The application of mass spectrometry-based techniques for the qualitative and quantitative analysis of the complex protein mixtures contained in most food preparations is playing a decisive role in the understanding of their nature, structure, functional properties and impact on human health. The application of mass spectrometry to protein analysis has been revolutionized in the recent years by the development of soft ionization techniques such as electrospray ionization and matrix assisted laser desorption/ionization, and by the introduction of multi-stage and ‘hybrid’ analyzers able to generate de novo amino acid sequence information. The interfacing of mass spectrometry with protein databases has resulted in entirely new possibilities of protein characterization, including the high sensitivity mapping (femtomole to attomole levels) of post-translational and other chemical modifications, protein conformations and protein–protein and protein–ligand interactions, and in general for proteomic studies, building up the core platform of modern proteomic science. MS-based strategies to food and nutrition proteomics are now capable to address a wide range of analytical questions which include issues related to food quality and safety, certification and traceability of (typical) products, and to the definition of the structure/function relationship of food proteins and peptides. These different aspects are necessarily interconnected and can be effectively understood and elucidated only by use of integrated, up-to-date analytical approaches. In this review, the main aspects of current and perspective applications of mass spectrometry and proteomic technologies to the structural characterization of food proteins are presented, with focus on issues related to their detection, identification, and quantification, relevant for their biochemical, technological and toxicological aspects.     

Ion mobility–mass spectrometry: a new paradigm for proteomics

Matrix-assisted laser desorption/ionization (MALDI) coupled with ion mobility–mass spectrometry (IM–MS) provides a rapid (μs–ms) means for the two-dimensional (2D) separation of complex biological samples (e.g., peptides, oligonucleotides, glycoconjugates, lipids, etc.), elucidation of solvent-free secondary structural elements (e.g., helices, β-hairpins, random coils, etc.), rapid identification of post-translational modifications (e.g., phosphorylation, glycosylation, etc.) or ligation of small molecules, and simultaneous and comprehensive sequencing information of biopolymers. In IM–MS, protein-identification information is complemented by structural characterization data, which is difficult to obtain using conventional proteomic techniques. New avenues for enhancing the figures of merit (e.g., sensitivity, limits of detection, dynamic range, and analyte selectivity) and optimizing IM–MS experimental parameters are described in the context of deriving new information at the forefront of proteomics research.     

Review of molecular modification techniques for improved detection of biomolecules by mass spectrometry

After a soft ionizing method was established, MS (mass spectrometry) has become a more common tool in biochemistry because soft ionization made it possible to detect large molecules such as proteins. Many kinds of applications were established to further utilize MS for the identification or quantitation of biomolecules. In this review, we introduce recent applications with special focus on chemical modification techniques and chemical probes developed for the MS determination of biomolecules.     

Proteolytic enzyme-immobilization techniques for MS-based protein analysis

Protein digestion utilizing proteases (e.g., trypsin, Lys C and other proteolytic enzymes) is one of the key sample-preparation steps in contemporary proteomics, followed by liquid chromatography coupled to mass spectrometry (MS). Tryptic digestion is traditionally performed in aqueous solutions, usually applying the enzyme and the sample in a 50:1 protein-to-protease ratio. Long digestion times (up to 24 h), auto-digestion sub-products and poor enzyme-to-substrate ratio are common issues with liquid-phase protein-digestion processes. The use of enzymes immobilized onto solid supports can minimize these problems by increasing enzyme-to-substrate ratios, significantly speeding up digestion times and reducing autolysis. The other main goal of protease immobilization is to obtain rugged, efficient enzyme reactors.In this article, we review the most important proteolytic enzyme-immobilization techniques with the main emphasis on fabrication of trypsin microreactors and nanoreactors and their utilization in bottom-up proteomics. We also discuss data reportedly obtained using the various immobilization protocols with respect to enzyme activity and MS-sequence coverage.     

应用双向电泳和质谱联用技术研究乳源蛋白的差异性

应用双向电泳和质谱联用技术,对不同乳源蛋白的差异性进行了研究。根据ImageMaster 2DPlatinum图像分析软件对不同乳源酪蛋白和乳清蛋白的双向电泳(2-DE)图谱进行蛋白斑点的匹配分析,获得21个存在于水牛奶中主要分布在低丰度蛋白区的酪蛋白差异蛋白点和24个存在于水牛奶中乳清蛋白差异蛋白点。这些差异蛋白点经质谱鉴定分析,得到4个属于水牛奶酪蛋白的主要组分和2个与水牛奶中酪蛋白有较高同源性的新组分,同时获得4个属于水牛奶乳清蛋白的主要组分和3个与水牛奶中乳清蛋白有较高同源性的组分。     

Novel UV assay for protein determination and the characterization of copper-protein complexes by mass spectrometry

A very simple, highly selective and sensitive assay of proteins based on the biuret absorption in the ultraviolet region has been developed. The well-known biuret assay is based on the reaction of proteins with copper ions under strong alkaline conditions to form a copper-protein complex. Yet, copper ions may seriously interfere with the determination if the measurement is made in the UV range. In the present approach, proteins mobilize copper ions from insoluble salts at different pH values, and the copper-protein complexes are investigated by UV spectrophotometry and mass spectrometry. Upon using copper phosphate, free copper ions do not interfere with the determination from 540 to 240 nm. Copper absorbance slowly increases from 240 to 190 nm where a blank with the reagents is recommended. A maximum absorbance for the copper-protein complex was found at 226 nm and high pH value. The stoichiometries of the copper-protein complexes measured directly with a mass spectrometer are pH dependent: half of the peptides without any histidine residue chelate just a single Cu²⁺ ion at pH 7.4 but each such peptide mobilizes from 1 to 6 Cu²⁺ ions at pH 10.3. To determine proteins, 1-1.5 ml of 1.8% KOH solution with 0-20 μg ml⁻¹ protein is treated with 25 mg of copper phosphate powder. The mixture is powerfully stirred, centrifuged, and the absorbance of the supernatant is measured at 226 nm in 1 cm quartz cuvettes against a blank of the reagents. The color system obeys Beer's law in the range 0.1-20 μg ml⁻¹ protein at this wavelength. The molar absorptivity value proved to be a characteristic of each protein being analyzed. Therefore, individual proteins should be used to plot calibration curves. This assay proved to be over 100 times more sensitive than the classical biuret procedure. The method is highly selective and the determination is little affected by the presence of other substances. All other important analytical parameters were studied and practical applicability of the method has been verified by the analysis of some biological materials.     

On-line cysteine modification for protein analysis: new probes for electrochemical tagging nanospray mass spectrometry

A series of electrogenerated selective electrophiles based on substituted benzoquinones has been characterized as tags for l-cysteine and cysteine residues in proteins. The electrophiles are generated electrochemically from the corresponding hydroquinones. It is shown from mass spectrometry analysis that the electrogenerated benzoquinone can tag the biomolecules. The rate constants pertaining to the addition of l-cysteine onto the electrogenerated benzoquinones have been determined using electrochemical techniques. The substitution patterns have been unraveled leading to the assessment of site-specific rate constants. It is shown that the rate constants are primarily dependent on the electronic nature of the substituents as expressed by the Hammett substitution constant. The apparent tagging yields observed for l-cysteine in nanospray mass spectrometry experiments do not correspond to the yields expected from the electrochemical study, as the ionisation efficiencies are highly dependent on the tag. Finally, the on-line tagging has been tested using β-lactoglobulin A and myoglobin. Based on these results, it is concluded that the tagging reaction is selective towards cysteine when it takes place in the nanospray interface. The results show that the methodology presented can be used for a rapid characterization and identification of reactive sites in biomolecules.     

Analysis of small amounts of solid samples by spark-source mass spectrometry

Various elements were determined in solid samples weighing < 2 mg by spark-source mass spectrometry. The samples were fixed on the top of a cylindrical graphite electrode using a conductive silver paint. After baking, the samples were sparked against a tantalum or gold wire. The method was used for the determination of impurities in steel, iron, molybdenum and CdSe. For samples weighing ca. 1 mg, detection limits of the order of 1 μg g^?1 were obtained. A relationship between the relative sensitivity factor and physical properties is proposed.     

Two-dimensional protein database of human pancreas

We report here the two-dimensional protein database of human pancreas. The proteins were analyzed by two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Totally, 302 proteins were identified, of which about 27% were enzymes with a broad range of catalytic activities. Several of these are specifically expressed in pancreas, such as pancreatic amylase, pancreatic stone protein, pancreatitis-associated protein, pancreatic lipase, pancreatic elastase, etc. Structural and cytoskeletal proteins are also strongly represented on the gels. Thus, the pancreatic proteome reflects the organ's function. This work paves the way for further studies on pancreatic protein expression in health and disease, such as diabetes and pancreatic cancer.     

Tryptic peptide analysis of protein binders in works of art by liquid chromatography-tandem mass spectrometry

A proteomics approach was used for the identification of protein binders in historical paints: the proteins were digested enzymatically into peptides using trypsin before being separated and detected by high performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS). Mascot (Matrix Science) was used to analyse the resulting data and for protein identification. In contrast to amino acid analysis, amino acid sequences could be studied that retain much more information about the proteins. The best extraction strategy was selected based on the number of peptides that were identified in the protein content of paint replicas using different methods. The influence of pigments on the extraction method was studied and the analytical characteristics of the selected method were determined. Finally this method was applied to historical paint microsamples on the anonymous early 15th century panel painting Crucifixion with St Catherine and St Barbara (Calvary of the Tanners), the St Catherine Altarpiece by Joes Beyaert (c. 1479) and two paintings by Pieter Brueghel the Younger (1617-1628).