Enrichment of low-molecular-weight proteins from biofluids for biomarker discovery

The dramatic progress in mass spectrometry-based methods of protein identification has triggered a new quest for disease-associated biomarkers. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and its variant surface-enhanced laser desorption/ionization mass spectrometry, provide effective means to explore the less studied information slice of the human serum proteome -- low-molecular-weight proteins and peptides. These low-molecular-weight proteins and peptides are promising for the detection of important biomarkers. Due to the significant experimental problems imposed by high-abundance and high-molecular-weight proteins, it is important to effectively remove these species prior to mass spectrometry analysis of the low-molecular-weight serum and plasma proteomes. In this review, the advantages afforded by recently introduced methods for prefractionation of serum, as they pertain to the detection and identification of biomarkers, will be discussed.     

Enrichment of low-molecular-weight proteins from biofluids for biomarker discovery

The dramatic progress in mass spectrometry-based methods of protein identification has triggered a new quest for disease-associated biomarkers. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and its variant surface-enhanced laser desorption/ionization mass spectrometry, provide effective means to explore the less studied information slice of the human serum proteome – low-molecular-weight proteins and peptides. These low-molecular-weight proteins and peptides are promising for the detection of important biomarkers. Due to the significant experimental problems imposed by high-abundance and high-molecular-weight proteins, it is important to effectively remove these species prior to mass spectrometry analysis of the low-molecular-weight serum and plasma proteomes. In this review, the advantages afforded by recently introduced methods for prefractionation of serum, as they pertain to the detection and identification of biomarkers, will be discussed.     

Rapid analysis of naphthalene and its metabolites in biological systems: Determination by high-performance liquid chromatography/fluorescence detection and by plasma desorption/chemical ionizations mass spectometry

A rapid procedure for the determination of naphthalene and its metabolites in bile of rainbow trout and mice is described. The integrated analytical techniques combine high-performance liquid chromatography/ultraviolet fluorescence detection and plasma desotption/chemical ionization mass spectrometry for identification and quantitation. After separation by reverse-phase liquid chromatography, naphthalene and its metablolites are detected and quantitated by ultraviolet fluoresence spectometry. Identification of two metabolites is confirmed by mass spectometry. A direct insertion probe tip for a conventional chemical ionization mass spectometer was modified to obtain spectra of thermally labile compounds. A spectrum of less than 100 ng of naphthyl glucuronide, a labile glucuronic acid conjugate of 1-naphthol, was obtained with this system.     

Dependence of the biological activity and mass spectrometric pattern on the structure peculiarities of the molecule of alkylating drug thiotepa

Structural and electronic parameters of the chemotherapeutic alkylating drug thiotepa obtained by MNDO and MINDO/3 quantum mechanical calculations are used to explain some physical, chemical and biological properties of this compound. On the basis of the revealed difference between the preferential conformations of the thiotepa molecule in crystal and vacuum, a conclusion is made concerning the precautions in the choice of the appropriate molecular geometry in the search of structure-activity correlations. The theoretical data are also applied to the explanation of some peculiarities of soft ionization mass spectra of thiotepa, in particular its sensitivity to high field effects and the absence of protonation. The modeling of some reactions of thiotepa directly in the conditions of field ionization mass spectrometric experiment is discussed.     

Utility of mass spectrometry for proteome ana lysis: part I. Conceptual and experimental approaches

This article is the first in a series of reviews intended as a tutorial providing the inexperienced, as well as the experienced, reader with an overview of principles of peptide and protein fragmentation in mass spectrometers for protein identification, surveying of the different types of instrument configurations and their combinations for protein identification. The first mass spectrometer was developed in 1899, but it took almost a century for the instrument to become a routine analytical method in proteomic research when fast atom bombardment ionization was developed, followed shortly by soft desorption/ionization methods, such as MALDI and electrospray ionization, to volatize biomolecules with masses of tens of kiloDaltons into the gas phase under vacuum pressure without destroying them. Thereafter, other soft ionization techniques that offered ambient conditions were also introduced, such as atmospheric pressure MALDI, direct analysis in real time, atmospheric-pressure solid analysis probe and hybrid ionization, sources of MALDI and electrospray ionization (e.g., two-step fused droplet electrospray ionization, laser desorption atmospheric-pressure chemical ionization, electrosonic spray ionization, desorption electrospray ionization, and electrospray-assisted laser desorption/ionization). The five basic types of mass analyzers currently used in proteomic research are the quadrupole, ion trap, orbitrap, Fourier transform ion cyclotron resonance and TOF instruments, which differ in how they determine the mass-to-charge ratios of the peptides. They have very different design and performance characteristics. These analyzers can be stand alone or, in some cases, put together in tandem or in conjunction with ion mobility mass spectrometry to take advantage of the strengths of each. Several singly or multiply charged fragment ion types, such as b, y, a, c, z, v, y and immonium ions are produced in the gas phase of the spectrometer. In the bottom-up sequencing approach for protein identification in a shotgun proteomic experiment, proteolytic digestion of proteins is accomplished by cleavage of the different bonds along the peptide backbone and/or side chain through a charge-directed transfer to the vicinity of the cleavage side. These various mass spectrometers and the types of ions produced have become important analytical tools for studying and analyzing proteins, peptides and amino acids.     

Rapid identification of DNA-binding proteins by mass spectrometry.

We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The determined molecular masses are often sufficient for identification. If not, the proteins are subjected to mass spectrometric peptide mapping followed by database searches. Apart from protein identification, the protocol also yields information on posttranslational modifications. The protocol was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA.     

The nucleotide-independent Fe(III)-binding site is located on beta subunit of the mitochondrial F(1)-ATPase

The need for methods to identify disease biomarkers is underscored by the survival-rate of patients diagnosed at early stages of cancer progression. Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a novel approach to biomarker discovery that combines two powerful techniques: chromatography and mass spectrometry. One of the key features of SELDI-TOF MS is its ability to provide a rapid protein expression profile from a variety of biological and clinical samples. It has been used for biomarker identification as well as the study of protein-protein, and protein-DNA interaction. The versatility of SELDI-TOF MS has allowed its use in projects ranging from the identification of potential diagnostic markers for prostate, bladder, breast, and ovarian cancers and Alzheimer's disease, to the study of biomolecular interactions and the characterization of posttranslational modifications. In this minireview we discuss the application of SELDI-TOF MS to protein biomarker discovery and profiling.     

Proteomic analysis of novel marine bacteria using MALDI and ESI mass spectrometry.

The objective of this study was to develop a mass spectrometric protocol to search for proteins related to phototrophy in marine bacteria. The genes that produce proteins involved in conversion of light into energy have been detected by cloning-sequencing from some of these bacteria, but it was previously unknown if these proteins were actually expressed. Attaining this study's goal was complicated by the fact that the samples consisted of miniscule cell pellets, which yielded small amounts of very complex mixtures of proteins. Sample preparation and analysis were tailored to optimize the probability of detecting the proteins of interest. It has been reported that using both matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) to analyze a mixture of peptides leads to the identification of more peptides that either technique alone. In order to exploit this complementarity between ESI and MALDI for proteomic analysis, samples were analyzed using both ionization techniques. With correct choices in sample preparation and ionization process, biologically relevant proteins can be identified out of small samples containing whole proteomes.     

Opportunities and limitations of SELDI-TOF-MS in biomedical research: practical advices

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, or surface-enhanced laser desorption/ionization ProteinChip technology, has been widely used in obtaining the quantitative profiles of tissue proteomes, particularly plasma proteomes. Its high-throughput nature and simplicity in its experimental procedures have allowed this technology to become a popular research tool for biomarker discovery in the past 5 years. After accumulating more research experiences, researchers now have a better understanding of the characteristics and limitations of this technology, as well as the pitfalls in biomarker research, by undertaking a comparative proteomic approach. This review provides an overview of the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, discusses its limitations and provides some possible solutions to help apply this technology to biomarker research.     

Applications of mass spectrometry in combinatorial chemistry

This article describes the use of two mass spectrometric techniques, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry, toward a variety of challenging problems in drug discovery and identification. Quantitative ESI was used to screen for inhibitor activity of two different enzymatic glycosylation reactions resulting in the identification of the most effective inhibitors and the determination of their IC50 (inhibitor concentration at 50% inhibition). Also described is a combinatorial extraction method used with automated MALDI mass spectrometry to improve upon the clinical analysis of the immunosuppressant drug cyclosporin A (CsA). Optimization was performed by generating an array of solvent systems which were screened (by MALDI-MS) for the most efficient extraction of CsA from whole blood. Ultimately a 70/30 hexane:CHCl3 mixture was identified as the most efficient binary solvent system for such extractions. In addition it was demonstrated that peptides and carbohydrates, covalently linked to a polymeric support (through a photolabile linker), can be directly analyzed by MALDI in a single step which requires no pretreatment of the sample to induce cleavage from the support. The UV laser light in the MALDI experiment was used to simultaneously promote the analyte's photolytic cleavage from the solid support and its gas phase ionization for subsequent mass spectral analysis. Overall, the strength of mass spectrometry lies in its versatility, making it a powerful analytical technique with which to characterize the diversity of compounds found in combinatorial libraries.     

Rapid and robust MALDI-TOF MS techniques for microbial identification: a brief overview of their diverse applications

in mass spectrometry have enabled the investigation of various biological systems by directly analyzing diverse sets of biomolecules (i.e., proteins, lipids, and carbohydrates), thus making a significant impact on the life sciences field. Over the past decade, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely utilized as a rapid and reliable method for the identification of microorganisms. MALDI-TOF MS has come into widespread use despite its relatively low resolving power (full width at half maximum, FWHM: < 5,000) and its incompatibility with tandem MS analysis, features with which other high-resolution mass spectrometers are equipped. Microbial identification is achieved by searching databases containing mass spectra of peptides and proteins extracted from microorganisms of interest, using scoring algorithms to match analyzed spectra with reference spectra. In this paper, we give a brief overview of the diverse applications of rapid and robust MALDI-TOF MS-based techniques for microbial identification in a variety of fields, such as clinical diagnosis and environmental and food monitoring. We also describe the fundamental principles of MALDI-TOF MS. The general specifications of the two major MS-based microbial identification systems available in the global market (BioTyper® and VITEK® MS Plus) and the distribution of these instruments in Republic of Korea are also discussed. The current review provides an understanding of this emerging microbial identification and classification technology and will help bacteriologists and cell biologists take advantage of this powerful technique.     

Improved protein identification through the use of unstained gels

In this work, a method for improved protein identification of low-abundance proteins using unstained gels, in combination with robotics and matrix-assisted laser desorption/ionization tandem mass spectrometry, has been developed and evaluated. Omitting the silver-staining process resulted in increased protein identification scores, an increase in the number of peptides observed in the MALDI mass spectrum, and improved quality of the tandem mass spectrometry data.     

Identification of proteins in cell-free extracts using liquid chromatography-electrospray ionization mass spectrometry

A rapid method for the identification and characterization of proteins in bacterial cell-free extracts has been developed using directly combined liquid chromatography-electrospray mass spectrometry. The usefulness of this technique is demonstrated for monitoring the expression and chemical modification of phosphoenolpyruvate-sugar phosphotransferase system (PTS) proteins from E. coli with molecular masses ranging from 9-65 kDa. The technique is characterized by minimal sample preparation, remarkable mass accuracy and resolution, reproducibility and the ability, unlike gel electrophoresis, to directly identify posttranslational modifications. The advantages of this technique over analogous matrix-assisted laser desorption ionization mass spectrometry approaches and its potential as a standard tool in the biomolecular research laboratory are discussed.     

Quantitative analysis of membrane proteins from breast cancer cell lines BT474 and MCF7 using multistep solid phase mass tagging and 2D LC/MS

We introduce a new multistep mass tagging technique and show its utility for reducing sample complexity when coupled with two-dimensional liquid chromatography/nano-electrospray ionization ion trap mass spectrometry (2D LC/nano ESI-MS). Solid-phase mass tagging reagents were used to identify and obtain relative quantitation of membrane proteins from two established breast cancer cell lines, BT474 and MCF7. The results presented in this study show that sample complexity can be reduced with corresponding increases in protein identification and quantitation.     

Colloidal Silver Staining of Electroblotted Proteins for High Sensitivity Peptide Mapping by Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

Mass spectrometric techniques for the identification of proteins either by amino acid sequencing or by correlation of mass spectral data with sequence databases are becoming increasingly sensitive and are rapidly approaching the limit of detection achieved by the staining of proteins in gels or, after electroblotting, on membranes. Here we present a technique for the sensitive staining of proteins electroblotted onto nitrocellulose or polyvinylidene difluoride membranes and enzymatic cleavage conditions for such proteins to achieve optimal recovery of peptides. The technique is based on the deposition of colloidal silver on the membrane-bound proteins. Peptide mixtures generated by proteolysis on the membrane were recovered at high yields and were compatible with analysis by reverse-phase chromatography and on-line electrospray ionization mass spectrometry. This simple and rapid colloidal silver staining procedure allowed the visualization of less than 5 ng of protein in a band and thus approached the sensitivity of silver staining in gels. We demonstrate that this method allows the detection of subpicomole amounts of electroblotted proteins and their identification by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry.     

Chemical ionization mass spectrometry of trimethylsilylated carbohydrates and organic acids retained in uremic serum

After appropriate sample pretreatment and derivatization, uremic serum was investigated by combined high resolution gas chromatography and mass spectrometry, using both electron impact and chemical ionization methods. Electron impact and chemical ionization spectra of a number of identified (trimethylsilylated) carbohydrates and organic acids are compared. The utilization of chemical ionization mass spectrometry, with isobutane as the reagent gas, is discussed in detail. The influence of the reagent gas pressure on the total ion current and on the spectral appearance was studied. The identification of compounds, based on electron impact mass spectral data, was confirmed and often aided appreciably by using this technique. The chemical ionization spectra of trimethylsilyated alditols and aldonic acids, as well as of other organic acids showed protonated molecular ions, whereas aldoses did not. Differences with electron impact spectra are found mainly in the high mass region. The loss of one or more trimethylsilanol groups becomes the predominating fragmentation route at higher reagent gas pressures.     

Quantitative profiling of plasma peptides in asthmatic mice using liquid chromatography and mass spectrometry

Asthma is increasing in prevalence worldwide as a result of factors associated with a Western lifestyle. However, simple and reliable diagnostic and prognostic markers are yet to be found. In an attempt to identify protein biomarker profiles among small molecular weight ranges, we employed an approach combining liquid chromatography with mass spectrometry, instead of two-dimensional gel electrophoresis (2-DE), which has previously been used to analyze protein expression patterns. Here we described its application to compare plasma peptides from control and chronic asthma mice. Peptides were quantitatively profiled as a multidimensional peptide mass fingerprint by a combination of reverse-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. They were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry. In this study, we quantitatively identified the fragment f of complement 3 (C3f), which is important in inflammation. C3f was significantly higher in controls than chronic asthma mice. Our strategy allowed the detection and identification of different plasma peptides between control and chronic asthma mice on a proteomic scale. Therefore, these results suggest that native small peptides detected by non-2-DE techniques may be useful and specific biomarkers of disease.     

基体辅助激光解吸电离质谱法及其应用

基体辅助激光解吸电离质谱是近几年才发展起来的一种新技术,它在生命科学研究中具有广阔的应用前景．对基体辅助激光解吸电离质谱技术的基本原理,运用基体辅助激光解吸电离质谱法研究蛋白质分子量的测定,蛋白质混合物的分离鉴定以及用基体辅助激光解吸电离质谱技术进行超快速的蛋白质序列分析和DNA序列分析的可行性和存在的问题作了介绍和讨论．     

Evaluation of parameters in peptide mass fingerprinting for protein identification by MALDI-TOF mass spectrometry

Protein identification by peptide mass fingerprinting, using the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), plays a major role in large proteome projects. In order to develop a simple and reliable method for protein identification by MALDI-TOF MS, we compared and evaluated the major steps in peptide mass fingerprinting. We found that the removal of excess enzyme from the in-gel digestion usually gave a few more peptide peaks, which were important for the identification of some proteins. Internal calibration always gave better results. However, for a large number of samples, two step calibrations (i.e. database search with peptide mass from external calibration, then the use of peptide masses from the search result as internal calibrants) were useful and convenient. From the evaluation and combination of steps that were already developed by others, we established a single overall procedure for peptide identification from a polyacrylamide gel.     

Precursor mass prediction by clustering ionization products in LC-MS-based metabolomics

Liquid chromatography-mass spectrometry (LC-MS) is becoming the dominant technology in metabolomics, involving the comprehensive analysis of small molecules in biological systems. However, its use is still limited mainly by challenges in global high-throughput identification of metabolites: LC-MS data is highly complex, particularly due to the formation of multiple ionization products from individual metabolites. To address the limitation in metabolite identification, we developed a principled approach, designed to exploit the multi-dimensional information hidden in the data. The workflow first clusters candidate ionization products of the same metabolite together which typically have similar retention time, then searches for mass relationships among them in order to determine their ion types and metabolite identity. The robustness of our approach was demonstrated by its application to the LC-MS profiles of cell culture supernatant, which accurately predicted most of the known media components in the samples. Compared to conventional methods, our approach was able to generate significantly fewer candidate metabolites without missing out valid ones, thus reducing false-positive matches. Additionally, improved confidence in identification is achieved since each prediction comes with a probable combination of known ion types. Hence, our integrative workflow provides precursor mass predictions with high confidence by identifying various ionization products which account for a large proportion of detected peaks, thus minimizing false positives.