Suppression of alpha-cyano-4-hydroxycinnamic acid matrix clusters and reduction of chemical noise in MALDI-TOF mass spectrometry

Progress in high-throughput MALDI-TOFMS analysis, especially in proteome applications, requires development of practical and efficient procedures for the preparation of proteins and peptides in a form suitable for high acquisition rates. These methods should improve successful identification of peptides, which depends on the signal intensity and the absence of interfering signals. Contamination of MALDI samples with alkali salts results in reduced MALDI peptide sensitivity and causes matrix cluster formation (widely reported for CHCA matrix) observed as signals dominating in the range below m/z 1200 in MALDI spectra. One way to remove these background signals, especially for concentrations of peptides lower than 10 fmol/microL, is to wash matrix/sample spots after peptide cocrystallization on the MALDI plate with deionized water prior to analysis. This method takes advantage of the low water solubility of the CHCA compared to its alkali salts. We report here that the application of some ammonium salt solutions, such as citrates and phosphates, instead of deionized water greatly improves the efficiency of this washing approach. Another way to reduce matrix cluster formation is to add ammonium salts as a part of the MALDI matrix. The best results were obtained with monoammonium phosphate, which successfully suppressed matrix clusters and improved sensitivity. Combining both of these approaches-the addition of ammonium salts in the CHCA matrix followed by one postcrystallization washing step with ammonium buffer-provided a substantial ( approximately 3-5-fold) improvement in the sensitivity of MALDI-MS detection compared to unwashed sample spots. This sample preparation method resulted in improved spectral quality and was essential for successful database searching for subnanomolar concentrations of protein digests.     

Characterization of Enterobacteria using MALDI-TOF mass spectrometry

A method is proposed for the rapid classification of Gram-negative Enterobacteria using on-slide solubilization and trypsin digestion of proteins, followed by MALDI-TOF MS analysis. Peptides were identified from tryptic digests using microsequencing by tandem mass spectrometry and database searches. Proteins from the outer membrane family (OMP) were consistently identified in the Enterobacteria Escherichia coli, Enterobacter cloacae, Erwinia herbicola, and Salmonella typhimurium. Database searches indicate that these OMP peptides observed are unique to the Enterobacteria order.     

Decomposition of ESR spectra using MALDI-TOF mass spectrometry

ESR (or EPR) spectroscopy on spin-labeled site-directed cysteine mutants is ideally suited for structural studies of membrane proteins due to its high sensitivity and its low demands with respect to sample purity and preparation. Many features can be inferred from the spectral line shape of an ESR spectrum, but the analysis of ESR spectra is complicated when multiple sites with different line shapes are present. Here, we present a method to decompose the spectrum of a doubly labeled peptide that is composed of a singly labeled, noninteracting component and a doubly labeled, dipolar-broadened component using a combination of optical and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The effect on the interspin distance calculation based on the dipolar broadening is quantified and discussed.     

Optimization of sample preparation for peptide sequencing by MALDI-TOF photofragment mass spectrometry

This paper describes the optimization of sample preparation for MALDI 193-nm photofragment ion time-of-flight mass spectrometry to sequence small to medium-sized peptides from peptide mixtures. We show that matrix additives, such as fructose and phenylbutyric acid have a dramatic effect on the abundance of fragment ions observed in the post-source decay spectra. A dried-droplet MALDI matrix consisting of 1:1 alpha-cyano-4-hydroxycinnamic acid/fructose proves to be an excellent matrix for photodissociation because [M + H]+ ions are formed with low internal energies, and the photofragment ion spectrum contains high abundances of sequence-informative ions. The addition of fructose appears to improve overall sample homogeneity and durability, as compared to conventional alpha-cyano-4-hydroxycinnamic acid dried-droplet preparations. MALDI-TOF photodissociation is then used to selectively sequence the peptides bradykinin (RPPGFSPFR), des-Arg9 bradykinin (RPPGFSPF), and substance P-amide (RPKPQQFFGLM-NH2) from a mixture of five peptides.     

Serum peptide profiling by magnetic particle-assisted, automated sample processing and MALDI-TOF mass spectrometry

Human serum contains a complex array of proteolytically derived peptides (serum peptidome) that may provide a correlate of biological events occurring in the entire organism; for instance, as a diagnostic for solid tumors (Petricoin, E. F.; Ardekani, A. M.; Hitt, B. A.; Levine, P. J.; Fusaro, V. A.; Steinberg, S. M.; Mills, G. B.; Simone, C.; Fishman, D. A.; Kohn, E. C.; Liotta, L. Lancet 2002, 359, 572-577). Here, we describe a novel, automated technology platform for the simultaneous measurement of serum peptides that is simple, scalable, and generates highly reproducible patterns. Peptides are captured and concentrated using reversed-phase (RP) batch processing in a magnetic particle-based format, automated on a liquid handling robot, and followed by a MALDI TOF mass spectrometric readout. The protocol is based on a detailed investigation of serum handling, RP ligand and eluant selection, small-volume robotics design, an optimized spectral acquisition program, and consistent peak extraction plus binning across a study set. The improved sensitivity and resolution allowed detection of 400 polypeptides (0.8-15-kDa range) in a single droplet (approximately 50 microL) of serum, and almost 2000 unique peptides in larger sample sets, which can then be analyzed using common microarray data analysis software. A pilot study indicated that sera from brain tumor patients can be distinguished from controls based on a pattern of 274 peptide masses. This, in turn, served to create a learning algorithm that correctly predicted 96.4% of the samples as either normal or diseased.     

Investigation of enzyme kinetics using quench-flow techniques with MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is combined off-line with rapid chemical quench-flow methods to investigate the pre-steady-state kinetics of a protein-tyrosine phosphatase (PTPase). PTPase kinetics are generally interrogated spectrophotometrically by the employment of an artificial, chromophoric substrate. However, that methodology places a constraint on the experiment, hampering studies of natural, biochemically relevant substrates that do not incorporate a chromophore. The mass spectrometric assay reported herein is based on the formation of a covalent phosphoenzyme intermediate during substrate turnover. This species is generated in the reaction regardless of the substrate studied and has a molecular weight 80 Da greater than that of the native enzyme. By following the appearance of this intermediate in a time-resolved manner, we can successfully measure pre-steady-state kinetics, regardless of the incorporation of a chromophore. The strengths of the mass-spectrometric assay are its uniform response to all substrates, simple and direct detection of covalent enzyme-substrate intermediates, and facile identification of enzyme heterogeneities that may affect enzymatic activity.     

Analysis of glycopeptides using lectin affinity chromatography with MALDI-TOF mass spectrometry

Glycopeptides prepared from 1 nmol of a mixture of glycoproteins, transferrin, and ribonuclease B by lysylendopeptidase digestion were isolated by lectin and cellulose column chromatographies, and then they were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and MALDI-quadrupole ion trap (QIT)-TOF mass spectrometry which enables the performance of MS ( n ) analysis. The lectin affinity preparation of glycopeptides with Sambucus nigra agglutinin and concanavalin A provides the glycan structure outlines for the sialyl linkage and the core structure of N-glycans. Such structural estimation was confirmed by MALDI-TOF MS and MALDI-QIT-TOF MS/MS. Amino acid sequences and location of glycosylation sites were determined by MALDI-QIT-TOF MS/MS/MS. Taken together, the combination of lectin column chromatography, MALDI-TOF MS, and MALDI-QIT-TOF MS ( n ) provides an easy way for the structural estimation of glycans and the rapid analysis of glycoproteomics.     

Structural characterization of oligosaccharides using MALDI-TOF/TOF tandem mass spectrometry

Extensive cross-ring fragmentation ions, which are very informative of the linkages of the monosaccharide residues constituting these molecules, were readily observed in the MALDI-TOF/TOF/MS/MS spectra of oligosaccharides. These ions, in some cases, were more intense than the commonly observed Y and B ions. The A-type ions observed for the simple oligosaccharides allowed the distinction between alpha(1-4)- and alpha(1-6)-linked isobaric structures. The distinction was based not merely on the differences in the type of ions formed, but also on the ion intensities. For example, both alpha(1-4)- and alpha(1-6)-linked isobaric structures produce ions resulting from the loss of approximately 120 m/z units, but with different intensities, as a result of the fact that they correspond to two different ions (i.e., 0,4A- and 2,4A-ions), requiring different energies to be formed. Abundant A- and X-type ions were also observed for high-mannose N-glycans, allowing the determination of linkages. In addition, the high resolution furnished by MALDI-TOF/TOF allowed determination of certain ions that were commonly overlooked by MALDI-TOF or MALDI-magnetic sector instruments as a result of their lower resolution. Moreover, as a result of the fact that MS/MS spectra for parent ions and all fragment ions are acquired under the same experimental conditions, accurate determination of the molar ratios of isomeric glycans in a mixture analyzed simultaneously by MALDI-TOF/TOF tandem MS becomes possible.     

Negative ion mass spectrometry of sialylated carbohydrates: discrimination of N-acetylneuraminic acid linkages by MALDI-TOF and ESI-TOF mass spectrometry

Negative ion MALDI and electrospray fragmentation spectra were recorded from 12 sialylated carbohydrates ranging from trisaccharides to biantennary N-linked glycans. D-Arabinosazone was found to be the most satisfactory MALDI matrix for these compounds. Fragmentation mechanisms were investigated with the aid of several synthesized analogues of the sugars labeled with 13C and 2H. The substitution position of the sialic acid (alpha2-->3 or alpha2-->6) was found to have a dramatic effect on the overall fragmentation pattern of these compounds, and several features of the spectra were identified that allowed the substitution pattern to be determined. In particular, the appearance of an ion at m/z 306 appeared to be diagnostic of the presence of an alpha2-->6-linked sialic acid. Selection and further fragmentation of the in-source (conevoltage) fragment ion corresponding to the trisaccharide Neu5Acalpha2-->3(or 6)Galbeta1-->4GlcNAc from larger, N-linked glycans, ionized by electrospray, gave fragmentation patterns identical to those of the reference trisaccharides, thus providing a method for confirming the sialic acid linkage.     

Identification of protein ligands in complex biological samples using intensity-fading MALDI-TOF mass spectrometry

The easy detection of biomolecular interactions in complex mixtures using a minimum amount of material is of prime interest in molecular and cellular biology research. In this work, a mass spectrometry MALDI-TOF based approach, which we call intensity-fading (IF MALDI-TOFMS), and which was designed for just such a purpose, is reported. This methodology is based on the use of the MALDI ion intensities to detect quickly the formation of complexes between nonimmobilized biomolecules in which a protein is one of the partners (protein-protein, protein-peptide, protein-organic molecule, and protein-nucleic acid complexes). The complex is detected through the decrease (fading) of the molecular ion intensities of the partners as directly compared to the MALDI mass spectrum of the mixture (problem and control molecules) following the addition of the target molecule. The potential of the approach is examined in several examples of model interactions, mainly involving small nonprotein and protein inhibitors of proteases, at both the qualitative and semiquantitative levels. Using this method, different protein ligands of proteolytic enzymes in total extracts of invertebrate organisms have been identified in a simple way. The proposed procedure should be easily applied to the high-throughput screening of biomolecules, opening a new experimental strategy in functional proteomics.     

Analysis of a model virus using residue-specific chemical cleavage and MALDI-TOF mass spectrometry

A nonenzymatic proteomics strategy is applied to the rapid identification of viruses. The approach provides solubilization and subsequent digestion of viral coat proteins in under 30 s. Acid digestions were carried out using a laboratory-quality microwave system equipped with temperature, pressure, and power controls, which allowed for precise optimization of experimental parameters. Under optimal conditions, this method provides an efficient alternative to traditional enzymatic digestion-based methods for virus identification. Following rapid microwave heating of a suspension of a model virus, RNA bacteriophage MS2, 13 chemical digestion products were detected in parallel with the coat protein precursor using MALDI-TOF MS. Because of the high sequence coverage obtained, the bacteriophage MS2 coat protein was identified with high confidence and the specificity of the identification allowed for the discrimination between bacteriophage MS2 and other closely related RNA bacteriophages.     

Analysis of viral glycoproteins by MALDI-TOF mass spectrometry

Membrane glycoproteins were shown to be useful biomarkers of enveloped viruses using on-target deglycosylation and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Sindbis virus, the prototype alpha-virus, was used as a model system. The glycoproteins and the capsid protein of the Sindbis virus were successfully detected by MALDI-TOF MS using two solvent systems. One of them is 0.5% n-octyl glucoside/0.5% trifluoroacetic acid. The two components of this solvent acted synergistically on the virus to help release and solubilize the structural proteins. The other is 70% acetonitrile/30% formic acid. This solvent solubilized the integral membrane glycoproteins very effectively even after serious aggregation. On-target deglycosylation was performed to confirm the detection of the glycoprotein peak and to produce protein moieties that can be used as biomarkers. After a simple and fast incubation using peptide-N-glycosidase F on target, sequential mass shifts were observed, which proved that the proteins detected at 51 000 Da have N-linked carbohydrate moieties at two sites. Observation of this mass shift could provide confirmatory evidence for viral identification.     

MALDI-TOF mass spectrometry analysis of amphipol-trapped membrane proteins

Amphipols (APols) are amphipathic polymers with the ability to substitute detergents to keep membrane proteins (MPs) soluble and functional in aqueous solutions. APols also protect MPs against denaturation. Here, we have examined the ability of APol-trapped MPs to be analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For that purpose, we have used ionic and nonionic APols and as model proteins (i) the transmembrane domain of Escherichia coli outer membrane protein A, a β-barrel, eubacterial MP, (ii) Halobacterium salinarum bacteriorhodopsin, an α-helical archaebacterial MP with a single cofactor, and (iii, iv) two eukaryotic MP complexes comprising multiple subunits and many cofactors, cytochrome b(6)f from the chloroplast of the green alga Chlamydomonas reinhardtii and cytochrome bc(1) from beef heart mitochondria. We show that these MP/APol complexes can be readily analyzed by MALDI-TOF-MS; most of the subunits and some lipids and cofactors were identified. APols alone, even ionic ones, had no deleterious effects on MS signals and were not detected in mass spectra. Thus, the combination of MP stabilization by APols and MS analyses provides an interesting new approach to investigating supramolecular interactions in biological membranes.     

Accurate molecular mass determination of mycolic acids by MALDI-TOF mass spectrometry

Mycolic acids, major and specific long-chain fatty (C70-C90) acid components of the mycobacterial cell envelope, were analyzed for the first time using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry operating in a reflectron mode. The various types of purified mycolates from representative mycobacterial species were analyzed using 2,5-DHB as matrix, because less than 10 pmol of mycolates was sufficient to obtain well-resolved mass spectra composed exclusively of pseudomolecular [M + Na]+ ions consistent with the structures deduced from the chemical analytical techniques applied to these molecules. Examination of the MALDI mass spectra demonstrated that the chain lengths of the various mycolates correlated with the growth rate of mycobacterial strains. Although slow growers, such as Mycobacterium tuberculosis and Mycobacterium ulcerans, produced a series of odd carbon numbers (C74-C82) of alpha-mycolic acids, rapid growers synthesized both odd and even carbon numbers. In addition, the main chain of oxygenated mycolic acids from slow growers were four to six carbon atoms longer than the corresponding alpha-mycolic acids, whereas rapid growers elaborated oxygenated homologues possessing the same chain lengths as their alpha-mycolic acids. Furthermore, a comparative analysis of the crude fatty acid mixtures from a wild-type strain of M. tuberculosis and its isogenic mutant effected in the synthesis of oxygenated mycolates by MALDI mass spectrometry revealed structural differences between the alpha-mycolates from the two strains. Thus, this technique appeared to be a rapid and highly sensitive technique for the analysis of mycolic acids, not only by providing accurate molecular masses and new structural information, but also by both reducing sample consumption and saving time.     

Oligosaccharide structures studied by hydrogen-deuterium exchange and MALDI-TOF mass spectrometry

Hydrogen-deuterium exchange matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (HX-MALDI-TOF MS) is reported for the determination of exchangeable protons in diverse oligosaccharide and glycoconjugate structures. The method has broad application for determining carbohydrate structure and conformation and to the study of carbohydrate-ligand interactions. The proton exchange process has been optimized to maximize the forward deuterium exchange and to suppress the well-known problem of back-exchange and is suitable for the analysis of all exchangeable proton types in carbohydrates. This has been validated for several diverse carbohydrate structures, including series of malto- and xylopyranose oligosaccharides; alpha- and beta-cyclodextrins; a nonreducing tetrasaccharide, stachyose; an N-acetylamide-containing oligosaccharide, chitotetraose; and a tertiary hydroxyl-containing antibiotic glycoconjugate, erythromycin.     

Microsequencing of glycans using 2-aminobenzamide and MALDI-TOF mass spectrometry: occurrence of unique linkage-dependent fragmentation

2-Aminobenzamide-derivatized oligosaccharides were separated by three lectin column chromatographies and then subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for structural characterization of the carbohydrates. The combination of sequential exoglycosidase digestion and MALDI-TOF MS greatly facilitates the monosaccharide sequencing and is more feasible than size-exclusion column chromatography in terms of the time consumed and the laboriousness of the procedure. By this strategy, microsequencing of 2-3 pmol of oligosaccharide derivatives could be achieved. Furthermore, spectra obtained by the post source decay (PSD) mode provide excellent sequence information. The relative intensities of metastable ions due to fragmentation at glycosidic linkages were different among linkage isomers of particular oligosaccharides. These results demonstrate that PSD analysis possesses significant potential for the estimation of glycosidic linkage in carbohydrate structures.     

The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer

A new matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometer is described for sequencing peptides. This instrument combines the advantages of high sensitivity for peptide analysis associated with MALDI and comprehensive fragmentation information provided by high-energy collision-induced dissociation (CID). Unlike the postsource decay technique that is widely used with MALDI-TOF instruments and typically combines as many as 10 separate spectra of different mass regions, this instrument allows complete fragment ion spectra to be obtained in a single acquisition at a fixed reflectron voltage. To achieve optimum resolution and focusing over the whole mass range, it may be desirable to acquire and combine three separate sections. Different combinations of MALDI matrix and collision gas determine the amount of internal energy deposited by the MALDI process and the CID process, which provide control over the extent and nature of the fragment ions observed. Examples of peptide sequencing are presented that identify sequence-dependent features and demonstrate the value of modifying the ionization and collision conditions to optimize the spectral information.     

Detection of plasmid insertion in Escherichia coli by MALDI-TOF mass spectrometry

A proteomics approach is reported for the rapid recognition of genetically modified Escherichia coli bacteria. The approach targets a class of proteins required for genetic manipulation of bacteria with plasmids and alleviates the need to construct extensive libraries of toxins and other predicted payload proteins. Detection was performed using MALDI-TOF MS to monitor peptide products after an on-probe enzymatic digestion. Digestion products were identified by searching their postsource decay spectra using MASCOT. A 5 min digestion time was required to observe peptide products from the genetic insert as well as the host bacterium. This proteomics approach enables rapid detection of genetic manipulation along with information about the host organism, both of which have forensic applications.     

Qualitative and quantitative analysis of pharmaceutical compounds by MALDI-TOF mass spectrometry

In this report, we discuss key issues for the successful application of MALDI-TOF mass spectrometry to quantify drugs. These include choice and preparation of matrix, nature of cationization agent, automation, and data analysis procedures. The high molecular weight matrix meso-tetrakis(pentafluorophenyl)porphyrin eliminates chemical noise in the low-mass range, a "brushing" spotting technique in combination with prestructured target plates enables fast preparation of homogeneous matrix crystals, and addition of Li+ leads to intense cationized drug species. Complex biological samples were cleaned up using a 96-well solid-phase extraction plate, and the purified samples were automatically spotted by a pipetting robot. To obtain a suitable data analysis procedure for the quantitative analysis of drugs by MALDI-TOF mass spectrometry, various data processing parameters were evaluated on our two model drugs lopinavir and ritonavir. Finally, and most importantly, it is shown that the above-described procedure can be successfully applied to quantify clinically relevant concentrations of lopinavir, an HIV protease inhibitor, in extracts of small numbers of peripheral blood mononuclear cells (1 x 10(6)).