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.     

Identification of disulfide-containing chemical cross-links in proteins using MALDI-TOF/TOF-mass spectrometry

Cross-linking can be used to identify spatial relationships between amino acids in proteins or protein complexes. A rapid and sensitive method for identifying the site of protein cross-linking using dithiobis(sulfosuccinimidyl propionate) (DTSSP) is presented and illustrated with experiments using murine cortactin, actin and acyl-CoA thioesterase. A characteristic 66 Da doublet, which arises from the asymmetric fragmentation of the disulfide of DTSSP-modified peptides, is observed in the mass spectra obtained under MALDI-TOF/TOF-MS conditions and allows rapid assignment of cross-links in modified proteins. This doublet is observed not only for linear cross-linked peptides but also in the mass spectra of cyclic cross-linked peptides when simultaneous fragmentation of the disulfide and the peptide backbone occurs. We suggest a likely mechanism for this fragmentation. We use guanidinylation of the cross-linked peptides with O-methyl isourea to extend the coverage of cross-linked peptides observed in this MALDI-MS technique. The methodology we report is robust and amenable to automation, and permits the analysis of native cystines along with those introduced by disulfide-containing cross-linkers.     

A programmable fragmentation analysis of proteins by in-source decay in MALDI-TOF mass spectrometry

Here we describe an algorithm for identifying peptides/ proteins of known sequence and unknown peptides from partial spectra generated by an in-source decay (ISD) technique coupled with matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. The identification of protein fragments is processed with a software program called CMATCH, which generates candidate subsequences for both known peptides/proteins and unknown peptides for the major product ions in the spectral range m/z 400-5000 and then matches these to known protein sequences contained in a reference database for the known peptides/proteins. CMATCH, which is compiled for MSDOS or WINDOWS95/NT, has two main advantages: first, the candidate subsequences are generated automatically without the need for supplementary information concerning the distribution of either N-terminal or C-terminal ions in the spectra for both known peptides/proteins and unknown peptides; second, the highest coordinated homologous sequences are picked up automatically from the reference database as the best matches with known peptides/proteins. Examples from the ISD spectra of several test proteins demonstrate the efficacy of this protein identification software.     

In-spray supercharging of peptides and proteins in electrospray ionization mass spectrometry

Enhanced charging, or supercharging, of analytes in electrospray ionization mass spectrometry (ESI MS) facilitates high resolution MS by reducing an ion mass-to-charge (m/z) ratio, increasing tandem mass spectrometry (MS/MS) efficiency. ESI MS supercharging is usually achieved by adding a supercharging reagent to the electrospray solution. Addition of these supercharging reagents to the mobile phase in liquid chromatography (LC)-MS/MS increases the average charge of enzymatically derived peptides and improves peptide and protein identification in large-scale bottom-up proteomics applications but disrupts chromatographic separation. Here, we demonstrate the average charge state of selected peptides and proteins increases by introducing the supercharging reagents directly into the ESI Taylor cone (in-spray supercharging) using a dual-sprayer ESI microchip. The results are comparable to those obtained by the addition of supercharging reagents directly into the analyte solution or LC mobile phase. Therefore, supercharging reaction can be accomplished on a time-scale of ion liberation from a droplet in the ESI ion source.     

Identification of biomarkers of whole Coxiella burnetii phase I by MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) specific biomarkers have been shown to be an effective tool for identifying microorganisms. In this study, we demonstrate the feasibility of using this technique to detect the obligate intracellular bacterium Coxiella burnetii, a category B bioterrorism agent. Specific biomarkers were detected in C. burnetii Nine Mile phase I (NMI) strain purified from embryonated egg yolk sac preparations. Whole organisms were applied directly to the MALDI target. MALDI-TOF MS analysis of C. burnetii NMI grown and purified at different times and places revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 1000-25000 Da. Statistical analysis of the averaged centroided masses uncovered at least 24 peptides or biomarkers. Three biomarkers observed in the MALDI-TOF MS spectrum consistently matched proteins that had been previously described in C. burnetii, one of them being the small cell variant protein A. MALDI-TOF MS analysis of whole organisms represents a sensitive and specific option for characterizing C. burnetii isolates, especially when coupled with antigen capture techniques. The method also has potential for several applications in basic microbial research, including regulation of gene expression.     

Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer

The present study reports a procedure developed for the identification of SDS-polyacrylamide gel electrophoretically separated proteins using an electrospray ionization quadrupole time-of-flight mass spectrometer (Q-TOF MS) equipped with pressurized sample introduction. It is based on in-gel digestion of the proteins without previous reduction/alkylation and on the capability of the Q-TOF MS to provide data suitable for peptide mass fingerprinting database searches and for tandem mass spectrometry (MS/MS) database searches (sequence tags). Omitting the reduction/alkylation step reduces sample contamination and sample loss, resulting in increased sensitivity. Omitting this step can leave disulfide-connected peptides in the analyte that can lead to misleading or ambiguous results from the peptide mass fingerprinting database search. This uncertainty, however, is overcome by MS/MS analysis of the peptides. Furthermore, the two complementary MS approaches increase the accuracy of the assignment of the unknown protein. This procedure is thus, highly sensitive, accurate, and rapid. In combination with pressurized nanospray sample introduction, it is suitable for automated sample handling. Here, we apply this approach to identify protein contaminants observed during the purification of the yeast DNA mismatch repair protein Mlh 1.     

On-line electrodialytic salt removal in electrospray ionization mass spectrometry of proteins

Salts and buffers, commonly used in isolation and stabilization of biological analytes, have a deleterious effect on electrospray ionization mass spectrometry (ESI-MS). Excessive concentrations of salts lead to ion suppression and adduct formation, which mask or complicate ion signals. In this work, we describe a salt remover (SR), configured as a three-compartment flow-through system, where the central compartment is separated from the outer compartments by a cation-exchange membrane (CEM) and an anion-exchange membrane (AEM). One platinum electrode is placed in each of the outer compartments, where water or electrolyte is flowing. The CEM electrode is held at a negative potential relative to the AEM side; cations/anions migrate by electrophoresis to the CEM/AEM receiver liquids, respectively. Proteins have poorer electrophoretic mobility relative to the buffer components, permitting removal of the salt. The salt-free proteins proceed to the ESI source. The capillary scale SR (internal volume 2.5 μL) described here effectively desalted continuous flows of NaCl solutions (200 mequiv/L at 1 μL/min, equivalent to a flux of 200 nequiv/min with 88% efficiency) and achieved >99.8% salt removal with 154 mM NaCl (isotonic saline) at 1 μL/min. With optimized current, >80% of concurrently present 20 μM protein was transmitted. Desalting efficiency and analyte loss was evaluated with different salt concentration and flow rate combinations under different applied current. Good-quality ESI-MS spectra of cytochrome c, myoglobin, and lysozyme as test proteins in a saline solution, passed through the SR, are demonstrated.     

Simultaneous quantification of human cardiac alpha- and beta-myosin heavy chain proteins by MALDI-TOF mass spectrometry

We have developed a novel method for quantifying protein isoforms, in both relative and absolute terms, based on MALDI-TOF mass spectrometry. The utility of the approach is demonstrated by quantifying the alpha and beta protein isoforms of myosin heavy chain (MyHC) in human atrial tissue. Alpha-MyHC (726-741) and beta-MyHC (724-739) were identified as isoform-specific tryptic peptides. A calibration curve was constructed by plotting ion current ratios against molar ratios of the two peptides prepared synthetically. MyHC was digested by trypsin and the ion current ratio determined for the two tryptic peptides. The ion current ratio was converted to the peptide ratio and hence the isoform ratio by reference to the standard curve. The accuracy of the method was confirmed by a comparison between these results and those determined by an established method of MyHC isoform ratio determination. So that the molar ratio could be converted to absolute values, a third peptide, an analogue of the two peptides being measured, was synthesized for use as an internal standard (IS). The measured ion current ratios of synthetic alpha-MyHC (726-741), beta-MyHC (724-739), and IS peptides were used to generate standard curves. A known quantity of the IS was added to the MyHC digests. The measured ion current ratios were converted to the actual quantities of the isoform-specific peptides and hence the actual quantity of each protein isoform by reference to the standard curves. This method is of general applicability, especially when isoform quantification is required.     

Evaluation of opiate separation by high-resolution electrospray ionization-ion mobility spectrometry/mass spectrometry

The separation of opiates and the primary metabolites was evaluated with ESI-IMS/MS. Seven opiate molecules were analyzed, and spectra were shown for each compound. The IMS separation of two isomers (morphine and norcodeine) was shown with baseline separation. Differences in the mobilities were found for the nonacetylated, monoacetylated, and biacetylated compounds. In this study, two primary findings are reported. First, IMS can easily separate metabolic isomers, and second, the two-dimensional separation capability of IMS/MS can be employed to confidently identify and separate both the opiates and metabolites. Although previous IMS studies have shown the separation of isomers, this is the first example to show the capability of IMS to separate metabolic isomers (within 70 s), a significant advantage in high-throughput screening for pharmacokinetic studies. Second, the monoacetylated and biacetylated compounds were found to form more compact ions for the sodium adducts in comparison to the protonated molecular ions. On the basis of the mobilities, information on structures and conformation can be deduced when sodium and protonated ions are compared.     

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.     

Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry

Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (microLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations. Conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin (Hb C, Hb E, Hb D-Los Angeles, Hb G-Philadelphia, Hb Hope, and Hb S). Hemoglobin proteins were isolated and purified, dehemed, (S)-carboxyami-domethylated, and then subjected to a combination proteolytic digestion to obtain a complex peptide mixture with multiple overlaps in sequence. Reversed-phase chromatographic separation of peptides was achieved on-line with MS utilizing a robust fritless microelectrospray interface. Tandem mass spectrometry was performed on an ion trap mass spectrometer using automated data-dependent MS/MS procedures. Tandem mass spectra were collected from the five most abundant ions in each scan using dynamic and isotopic exclusion to minimize redundancy. The spectra were analyzed by a version of the SEQUEST algorithm modified to identify amino acid substations resulting from SNPs.     

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.     

Zeptomole-sensitivity electrospray ionization--Fourier transform ion cyclotron resonance mass spectrometry of proteins

Methods are being developed for ultrasensitive protein characterization based upon electrospray ionization (ESI) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The sensitivity of a FTICR mass spectrometer equipped with an ESI source depends on the overall ion transmission, which combines the probability of ionization, transmission efficiency, and ion trapping in the FTICR cell. Our developments implemented in a 3.5 tesla FTICR mass spectrometer include introduction and optimization of a newly designed electrodynamic ion funnel in the ESI interface, improving the ion beam characteristics in a quadrupole-electrostatic ion guide interface, and modification of the electrostatic ion guide. These developments provide a detection limit of approximately 30 zmol (approximately 18,000 molecules) for proteins with molecular weights ranging from 8 to 20 kDa.     

Compositional analysis of glycosaminoglycans by electrospray mass spectrometry

The purpose of this work is to analyze glycosaminoglycans (GAGs) directly from complex mixtures without the need to purify individual components. Novel conditions for negative ion electrospray MS of chondroitin sulfate (CS) oligosaccharides are described in which sodium adduction and fragmentation are avoided. Differentiation between positional sulfation isomers is demonstrated for CS disaccharides, and a selected reaction monitoring scheme is used to quantify sulfation isomers in disaccharides liberated from decorin and biglycan. A size exclusion chromatography LC/MS method is shown to be effective for compositional analysis of longer CS oligosaccharides. The SEC step serves to simplify the composition of GAGs entering the mass spectrometer at any time, thus allowing the masses of the constituent molecules to be extracted. Mass spectrometric detection produces far more information than conventional UV or fluorescent detectors and allows the monosaccharide composition of individual components to be determined.     

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.     

Identification and quantification of arsenolipids using reversed-phase HPLC coupled simultaneously to high-resolution ICPMS and high-resolution electrospray MS without species-specific standards

Although it has been known for decades that arsenic forms fat-soluble arsenic compounds, only recent attempts to identify the compounds have been successful by using a combination of fractionation and elemental and molecular mass spectrometry. Here we show that arsenolipids can directly be identified and quantified in biological extracts using reversed-phase high-performance liquid chromatography (RP-HPLC) simultaneously online-coupled to high-resolution inductively coupled plasma mass spectrometry (ICPMS) and high-resolution electrospray mass spectrometry (ES-MS) without having a lipophilic arsenic standard available. Using a methanol gradient for the separation made it necessary to use a gradient-dependent arsenic response factor for the quantification of the fat-soluble arsenic species in the extract. The response factor was obtained by using the ICPMS signal of known concentration of arsenic. The arsenic response was used to determine species-specific response factors for the different arsenic species. The retention time for the arsenic species was utilized to mine the ES-MS data for accurate mass and their tandem mass spectrometry (MS/MS) fragmentation pattern to give information of molecular formula and structure information. The majority of arsenolipids, found in the hexane phase of fish meal from capelin ( Mallotus villosus ) was in the form of three dimethylarsinoyl hydrocarbons (C(23)H(38)AsO, C(17)H(38)AsO, C(19)H(42)AsO) with minor amounts of dimethylarsinoyl fatty acids (C(17)H(36)AsO(3), C(23)H(38)AsO(3), C(24)H(38)AsO(3)). One of the dimethylarsinoyl fatty acids (C(24)H(38)AsO(3)), with an even number of carbon in the fatty acid chain, was identified for the first time in this work. This molecular formula is unusual and in contrast to all previously identified arsenic-containing fatty acids with odd numbers of carbon.     

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)).     

Analysis of nucleic acids by capillary ion-pair reversed-phase HPLC coupled to negative-ion electrospray ionization mass spectrometry

Ion-pair reversed-phase high-performance liquid chromatography was successfully coupled to negative-ion electrospray ionization mass spectrometry by using 60 × 0.20 mm i.d. capillary columns packed with 2.3-μm micropellicular, octadecylated poly(styrene/divinylbenzene) particles as stationary phase and gradients of acetonitrile in 50 mM aqueous triethylammonium bicarbonate as mobile phase. Systematic variation of the eluent composition, such as concentration of ion-pair reagent, anion in the ion-pair reagent, solution pH, and acetonitrile concentration led to the conclusion that most parameters have opposite effects on chromatographic and mass spectrometric performances. The use of acetonitrile as sheath liquid enabled the rapid and highly efficient separation and detection of phosphorylated and nonphosphorylated oligonucleotides ranging in size from 8 to 40 nucleotides. High-quality full-scan mass spectra showing little cation adduction were acquired from which the molecular masses of the separated oligonucleotides were calculated with an accuracy of 0.011%. With calibration curves being linear over at least 2 orders of magnitude, the lower limits of detection for a oligodeoxythymidine 16-mer were 104 fmol with full scan and 710 amol with selected-ion-monitoring data acquisition. The potential of ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry was demonstrated for mixed-sequence oligomers by the characterization of a reaction mixture from solid-phase synthesis of a 40-mer oligonucleotide.     

Identification of proteins in complexes by solid-phase microextraction/multistep elution/capillary electrophoresis/tandem mass spectrometry.

A method to directly identify proteins in complex mixtures by solid-phase microextraction (micro-SPE)/multistep elution/capillary electrophoresis (CE)/tandem mass spectrometry (MS/MS) is described. A sheathless liquid-metal junction interface is used to interface CE and electrospray ionization MS/MS. A subfemtomole detection limit is achieved for protein identification through database searching using MS/MS data. The SPE serves as a semiseparation dimension using an organic-phase step-elution gradient in combination with the second separation dimension for increased resolving power of complex peptide mixtures. This approach improves the concentration detection limit for CE and allows more proteins in complex mixtures to be identified. A 75-protein complex from yeast ribosome is analyzed using this method and 80-90% of the proteins in the complex can be identified by searching the database using the MS/MS data from a complete analysis. This multidimensional CE/MS/MS methodology provides an alternative to multidimensional liquid chromatography/MS/MS for direct identification of small amounts of protein in mixtures.