An exponential dilution gradient system for nanoscale liquid chromatography in combination with MALDI or Nano-ESI mass spectrometry for proteolytic digests

A simple and inexpensive nano high performance liquid chromatography system (nano-LC) employing the exponential dilution method for gradient separations was built. The system was used to analyze a tryptic digest of Escherichia coli uracil DNA glycosylase (Ung; Mr = 25,563), a DNA-binding protein that initiates the uracil-excision DNA repair process by catalyzing the release of uracil from the deoxyribose phosphate backbone of DNA. Both on-line and off-line approaches to analyzing peptides produced by in-gel digestion of Ung are demonstrated. The on-line approach uses nano-high performance liquid chromatography (HPLC)/micro-electrospray MS to assign peptide masses. The off-line approach uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nano-electrospray/collision-induced dissociation (CID) tandem mass spectrometry, to analyze fractions (2-3 microL) collected manually from the nano-LC system. The nano-electrospray technique allows detailed fragmentation information to be obtained at different collision energies with only a marginal increase in sample handling due to the nano-LC step.     

Alkaline liquid chromatography/electrospray ionization skimmer collision-induced dissociation mass spectrometry for phosphopeptide screening.

A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS). Phosphorylation-specific marker ions (m/z 79, PO(3)(-), and m/z 97, H(2)PO(4)(-)) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO(3)(-)) and m/z 97 (H(2)PO(4)(-)) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C(2)F(3)O(-) fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic beta-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by (mu)LC/ESI-sCID-MS and (mu)LC/ESI-MS analysis.     

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix-assisted laser desorption/ionization and electrospray-mass spectrometry detection

High throughput, outstanding certainty in peptide/protein identification, exceptional resolution, and quantitative information are essential pillars in proteome research. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has proven to meet these requirements. Soft ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), have paved the way for the story of success of CE-MS in the analysis of biomolecules and both approaches are subject of discussion in this article. Meanwhile, CE-MS is far away from representing a homogeneous field. Therefore the review will cover a vast area including the coupling of different modes of CE (capillary zone electrophoresis, capillary isoelectric foscusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as on-line preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) applied to compensate for restricted detection sensitivity. Special attention is given to improvements in interfacing, namely addressing nanospray and coaxial sheath liquid design. Peptide mapping, collision-induced dissociation with subsequent tandem MS, and amendments in mass accuracy of instruments improve information validity gained from MS data. With 2-D on-line coupling of liquid chromatography (LC) and CE a further topic will be discussed. A special section is dedicated to recent attempts in establishing CE-ESI-MS in proteomics, in the clinical and diagnostic field, and in the food sector.     

Quantitative determination of piritramide in human plasma and urine by off- and on-line solid-phase extraction liquid chromatography coupled to tandem mass spectrometry

A method for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) quantification of piritramide, a synthetic opioid, in plasma after conventional off-line solid-phase extraction (SPE) and in urine by on-line SPE-LC/MS/MS in positive electrospray mode was developed and validated. Applicability of the on-line approach for plasma samples was also tested. Deuterated piritramide served as internal standard. For the off-line SPE plasma method mixed cation-exchange SPE cartridges and a 150 x 2 mm C18 column with isocratic elution were used. For the on-line SPE method, a Waters Oasis HLB extraction column and the same C18 analytical column in a column-switching set-up with gradient elution were utilized. All assays were linear within a range of 0.5-100 ng/mL with a limit of detection of 0.05 ng/mL. The intra- and interday coefficients of variance ranged from 1.3 to 6.1% for plasma and 0.5 to 6.4% for urine, respectively. The extraction recovery for the off-line plasma assay was between 90.7 and 100.0%. Influence of matrix effects, and freeze/thaw and long-term stability were validated for both approaches; influence of urine pH additionally for quantification in urine.     

Identification of peptides in antimicrobial fractions of cheese extracts by electrospray ionization ion trap mass spectrometry coupled to a two-dimensional liquid chromatographic separation

Electrospray ionization ion trap mass spectrometry (ESI-ITMS) coupled to a two-dimensional liquid chromatographic separation was applied to the identification of peptides in antimicrobial fractions of the aqueous extracts of nine Italian cheese varieties. In particular, the chromatographic fractions collected during a preliminary fast protein liquid chromatography (FPLC) separation on the cheese extracts were assayed for antimicrobial activity towards Lactobacillus sakei A15. Active fractions were subsequently analyzed by reversed-phase high-performance liquid chromatography electrospray ionization sequential mass spectrometry (HPLC/ESI)-ITMSn, with n up to 3. Peptide identification was then performed starting from a conventional proteomics approach based on tandem mass spectrometric (MS/MS) analysis followed by database searching. In many cases this strategy had to be integrated by a careful correlation between spectral information and predicted peptide fragmentation, in order to reach unambiguous identifications. When even this integrated approach failed, MS3 measurements provided decisive information on the amino acid sequence of some peptides, through fragmentation of pendant groups along the peptide chain. As a result, 45 peptides, all arising from hydrolysis of milk caseins, were identified in nine antimicrobial FPLC fractions of aqueous extracts obtained from five of the nine cheese varieties considered. Many of them corresponded to peptides already known to exhibit biological activity.     

A novel software tool for copolymer characterization by coupling of liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The results of copolymer characterization by coupling of chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques and subsequent calculation of copolymer composition using a novel software tool 'MassChrom2D' are presented. For high-resolution mass analysis copolymer samples were fractionated by means of liquid adsorption chromatography (LAC). These fractions were investigated off-line by MALDI-TOF MS. Various mono-n-butyl ethers of polyethylene oxide-polypropylene oxide copolymers (PEO-co-PPO) were investigated. As well as the copolymer composition presented in two-dimensional plots, the applied approach can give additional hints on specific structure-dependent separation conditions in chromatography.     

Alteration of protein composition in mouse thymocytes by signals through T-cell receptor

To avoid destructive autoimmunity, T-cell precursors (thymocytes) expressing autoreactive T-cell receptor are deleted in the thymus via an apoptotic process by the signals from the T-cell receptor-CD3 complexes. In order to analyze the apoptotic mechanism, we established a cell-free system using the lysates from mouse thymocytes treated in vivo with anti-CD3 monoclonal antibody (mAb). The soluble cytosolic high molecular mass protein fraction from the anti-CD3-treated thymocytes revealed an activity that directly induces nuclear apoptotic morphological changes and DNA fragmentation. This fragmentation activity was not observed in the fraction from the thymocytes without anti-CD3 treatment. Proteins in both fractions were separated by two-dimensional electrophoresis. The silver-stained gels revealed differences in protein spots. These protein spots were identified by database searching of mass spectrometric (MS) and tandem mass spectrometric (MS/MS) data obtained from in-gel tryptic digests of the spots, using an integrated system of liquid chromatography/electrospray ionization/ion-trap mass spectrometry. In this study, the high mobility group protein HMG2 was identified as one of the cytosolic proteins that is increased by the signals from the T-cell receptor, and heterogeneous nuclear ribonucleoprotein A2/B1 and glyceraldehyde 3-phosphate dehydrogenase were found to be decreased by the signals.     

EJMS protocol: systematic studies on TiO2-based phosphopeptide enrichment procedures upon in-solution and in-gel digestions of proteins. Are there readily applicable protocols suitable for matrix-assisted laser desorption/ionization mass spectrometry-based phosphopeptide stability estimations?

There have been many successful efforts to enrich phosphopeptides in complex protein mixtures by the use of immobilized metal affinity chromatography (IMAC) and/or metal oxide affinity chromatography (MOAC) with which mass spectrometric analysis of phosphopeptides has become state of the art in specialized laboratories, mostly applying nanoLC electrospray ionization mass spectrometry-based investigations. However, widespread use of these powerful techniques is still not achieved. In this study, we present a ready-to-use phosphopeptide enrichment procedure using commercially available TiO(2)-loaded pipette tips in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses. Using α-casein as a model protein and citric acid as additive during sample loading, a similar enrichment success can be achieved as compared to applying 2,5- dihydroxy benzoic acid (DHB) for this task. But the DHB-inherited drawbacks are eliminated. In addition, we show that combining DHB and 2,4,6-trihydroxy acetophenone (THAP) as matrix for MALDI-MS measurements retains the sensitivity of DHB for phosphopeptide analysis but adds the homogenous crystallization properties of THAP, enabling preparation of evenly distributed matrix surfaces on MALDI-MS anchor targets, a prerequisite for automated MALDI- MS analyses. Tripartite motif-containing protein 28 and stathmin are two examples for which successful phosphopeptide enrichment of either sodium dodecyl sulfate polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis-separated proteins is shown. Finally, high resolution MALDI Fourier transform ion cyclotron resonance mass spectrometry after phosphopeptide enrichment suggests that chemical dephosphorylation may occur as a side reaction during basic elution of phosphopeptides bound to MOAC surfaces, suggesting that proteome-wide phosphopeptide analyses ought to be interpreted with caution. In contrast, in-depth analysis of phosphopeptide/non-phosphorylated peptide siblings may be used to estimate stability differences of phosphorylation sites in individual proteins, possibly adding valuable information on biological regulation processes.     

A fast screening method for the identification of siderophores from fluorescent Pseudomonas spp. by liquid chromatography/electrospray mass spectrometry

A screening method was developed for the fast identification of known pyoverdin-type siderophores produced by fluorescent Pseudomonas spp. It is based on reversed-phase high-performance liquid chromatography interfaced with electrospray ionization mass spectrometry of the Sep-Pak RP-C18 culture supernatant extracts. The siderophores of five bacterial strains were characterized by their molecular masses obtained from their doubly protonated molecular ions [M + 2H]2+ and their UV/visible spectra recorded with a diode-array detector. Additional structural information was gained by skimmer collision-induced dissociation experiments. For all strains new minor siderophores were found. A table of fully or partially identified pyoverdins and related siderophores is provided which will be the basis for screening studies.     

Fourier transform ion cyclotron resonance mass spectrometry of covalent adducts of proteins and 4-hydroxy-2-nonenal, a reactive end-product of lipid peroxidation

Covalent adduction of the model protein apomyoglobin by 4-hydroxy-2-nonenal, a reactive end-product of lipid peroxidation, was characterized by nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The high mass resolving power and mass measurement accuracy of the instrument facilitated a detailed compositional analysis of the complex reaction product without the need for deconvolution and transformation to clearly show the pattern of adduction and component molecular weights. Our study has also demonstrated the value of electron capture dissociation over collision-induced dissociation for the tandem mass spectrometric determination of site modification for the 4-hydroxy-2-nonenal adduct of oxidized insulin B chain as an example. Figure FTICR allowed characterization of 4-hydroxy-2-nonenal (HNE)-modified apomyoglobin (an expanded spectrum of the +15 charge state is shown)     

Top-down identification of endogenous peptides up to 9 kDa in cerebrospinal fluid and brain tissue by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry

Recent work on protein and peptide biomarker patterns revealed the difficulties in identifying their molecular components, which is indispensable for validation of the biological context. Cerebrospinal fluid and brain tissue are used as sources to discover new biomarkers, e.g. for neurodegenerative diseases. Many of these biomarker candidates are peptides with a molecular mass of <10 kDa. Their identification is favourably achieved with a 'top-down' approach, because this requires less purification and an enzymatic cleavage will often not yield enough specific fragments for successful database searches. Here, we describe an approach using quadrupole time-of-flight mass spectrometry (TOFMS) as a highly efficient mass spectrometric purification and identification tool after off-line decomplexation of biological samples by liquid chromatography. After initial peptidomic screening with matrix-assisted laser desorption/ionization (MALDI) TOFMS, the elution behaviour in chromatography and the exact molecular mass were used to locate the same signals in nanoelectrospray measurements. Most of the peaks detected in MALDI-TOFMS could be retrieved in nanoelectrospray quadrupole TOFMS. Suitable collision energies for informative fragment spectra were investigated for different parent ions, charge states and molecular masses. After collision-induced dissociation, the resulting fragmentation data of multiply charged ions can become much more complicated than those derived from tryptic peptide digests. However, the mass accuracy and resolution of quadrupole TOF instruments results in high-quality data suitable for determining peptide sequences. The protein precursor, proteolytic processing and post-translational modifications were identified by automated database searches. This is demonstrated by the exemplary identifications of thymosin beta-4 (5.0 kDa) and NPY (4.3 kDa) from rat hypothalamic tissue and ubiquitin (8.6 kDa) from human cerebrospinal fluid. The high data quality should also allow for de novo identification. This methodology is generally applicable for peptides up to a molecular mass of about 10 kDa from body fluids, tissues or other biological sources.     

Enhanced characterization of singly protonated phosphopeptide ions by femtosecond laser-induced ionization/dissociation tandem mass spectrometry (fs-LID-MS/MS)

To develop an improved understanding of the regulatory role that post-translational modifications (PTMs) involving phosphorylation play in the maintenance of normal cellular function, tandem mass spectrometry (MS/MS) strategies coupled with ion activation techniques such as collision-induced dissociation (CID) and electron-transfer dissociation (ETD) are typically employed to identify the presence and site-specific locations of the phosphate moieties within a given phosphoprotein of interest. However, the ability of these techniques to obtain sufficient structural information for unambiguous phosphopeptide identification and characterization is highly dependent on the ion activation method employed and the properties of the precursor ion that is subjected to dissociation. Herein, we describe the application of a recently developed alternative ion activation technique for phosphopeptide analysis, termed femtosecond laser-induced ionization/dissociation (fs-LID). In contrast to CID and ETD, fs-LID is shown to be particularly suited to the analysis of singly protonated phosphopeptide ions, yielding a wide range of product ions including a, b, c, x, y, and z sequence ions, as well as ions that are potentially diagnostic of the positions of phosphorylation (e.g., ‘a _n+1–98’). Importantly, the lack of phosphate moiety losses or phosphate group ‘scrambling’ provides unambiguous information for sequence identification and phosphorylation site characterization. Therefore, fs-LID-MS/MS can serve as a complementary technique to established methodologies for phosphoproteomic analysis.     

Arsenosugar identification in seaweed extracts using high-performance liquid chromatography/electrospray ion trap mass spectrometry

The development of analytical techniques suitable for providing structural information on a wide range of elemental species is a growing necessity. For arsenic speciation a variety of mass spectrometric techniques, mainly inductively coupled plasma mass spectrometry (ICP-MS) and electrospray tandem mass spectrometry (ES-MS/MS) coupled on-line with high-performance liquid chromatography (HPLC), are in use. In this paper we report the identification of arsenic species present in samples of marine origin (seaweed extracts) using ES ion trap mass spectrometry (IT) multistage mass spectrometry (MS(n)). Both reversed-phase and anion-exchange HPLC have been coupled on-line to ES-ITMS. Product ion scans with multiple stages of tandem MS (MS(n); n=2-4) were used to acquire diagnostic data for each arsenosugar. The spectra contain structurally characteristic fragment ions for each of the arsenosugars examined. In addition it was observed that upon successive stages of collision-induced dissociation (CID) a common product ion (m/z 237) was formed from all four arsenosugars examined. This product ion has the potential to be used as an indicator for the presence of dimethylated arsenosugars (dimethylarsinoylribosides). The HPLC/ES-ITMS(n) method developed allows the sensitive identification of arsenosugars present in crude seaweed extracts without the need for extended sample preparation. In fact, sample preparation requirements are identical to those typically employed for HPLC/ICP-MS analysis. Additionally, the resulting product ions are structurally diagnostic of the arsenosugars examined, and tandem mass spectra are reproducible and correspond well to those obtained using other low-energy CID techniques. As a result, the HPLC/ES-ITMS(n) approach minimises the potential for arsenic species misidentification and has great potential as a means of overcoming the need for characterised standards.     

Structural characterization of complex bacterial glycolipids by Fourier transform mass spectrometry

Bacterial glycolipids are complex amphiphilic molecules which are on the one hand of utmost importance for the organization and function of bacterial membranes, and which on the other hand play a major role in the activation of cells of the innate and adaptive immune system of the host. Already small alterations of their chemical structure may influence the biological activity tremendously. Due to their intrinsic biological heterogeneity [number and type of fatty acids, saccharide structures, and substitution with e.g. phosphate (P), 2-aminoethyl- (pyro)phosphate groups (P-Etn) or 4-amino-4-deoxyarabinose (Ara4N)], separation of the different components are a prerequisite for unequivocal chemical and NMR structural analyses. In this contribution the structural information which can be obtained from heterogeneous samples of glycolipids by Fourier transform (FT) ion cyclotron resonance mass spectrometric methods is described. By means of recently analysed complex biological samples the possibilities of high resolution electrospray ionization FT-MS are demonstrated. Capillary skimmer dissociation, as well as tandem mass spectrometry MS/MS analysis utilizing collision-induced dissociation and infrared multiphoton dissociation, are compared and their advantages to provide structural information of diagnostic importance are discussed.     

Ion/molecule reactions with dimethyl ether and dimethyl-d6 ether: differentiation of four isomers contained in patchouli essential oil

In this study, we show that it is possible to differentiate four sesquiterpene isomers (C(15)H(24)) preliminarily separated by gas chromatography/mass spectrometry (GC/MS). Dimethyl ether is evaluated as a selective ionization reagent and the relative abundance of adducts formed with this reagent gas under positive chemical ionization conditions are compared and adduct ions are characterized using collision-induced dissociation. The mechanisms have been confirmed by achieving the same experiments with deuterated dimethyl ether. Copyright 2000 John Wiley & Sons, Ltd.     

Viscosalines B(1,2) and E(1,2): challenging new 3-alkyl pyridinium alkaloids from the marine sponge Haliclona viscosa

Four new 3-alkyl pyridinium alkaloids, the viscosalines?B(1) (1?a), B(2) (1?b), E(1) (2?a), and E(2) (2?b), were isolated from the Arctic sponge Haliclona viscosa. The structure elucidation of these isomeric compounds was challenging due to ambiguous fragments that derive during "standard" mass spectrometric fragmentation experiments. The final structure elucidation relied on the use of a combination of synthesis, liquid chromatography, and mass spectrometry. Three different mass spectrometers were used to differentiate between the synthetic structural isomers: a time-of-flight (TOF) mass spectrometer and two ion-trap mass spectrometers with different ion-transfer technologies (i.e., skimmer versus funnel optics). Although at first none of the spectrometers returned spectra that permitted structure elucidation, all three mass spectrometers provided analysis that successfully differentiated between the isomers after thorough method optimization. The use of in-source collision-induced dissociation (CID) with the ion trap and TOF instrument returned the most interesting results. The mode of fragmentation of the viscosalines under different experimental conditions is described herein. After successful optimization of the mass spectrometric method applied, the chromatographic method was improved to distinguish the previously inseparable isomers. Finally, both the liquid chromatography and mass spectrometric methods were applied to the natural products and the results compared to those from the synthetic compounds.     

Mass spectrometric identification of in vivo carbamylation of the amino terminus of Ectothiorhodospira mobilis high-potential iron-sulfur protein,isozyme 1

The complete amino acid sequence of a novel high-potential iron-sulfur protein (HiPIP) isozyme 1 from the moderately halophilic phototrophic bacterium Ectothiorhodospira mobilis was determined by a combined approach of chemical and mass spectrometric sequencing techniques. By mass analysis of the apo- and holo-protein in the positive electrospray ionization mode using different electrospray solvents, the protein was found to be post-translationally modified by a moiety of 43 Da. Further analysis showed the nature and location of this modification to be a carbamyl group at the N-terminus of the HiPIP. This rare type of modification has previously been reported to occur in the water-soluble human lens alphaB-crystallin, class D beta-lactamases and some prokaryotic ureases, albeit at an internal lysine residue. In this paper, we discuss the mass spectrometric features of a carbamylated residue at the N-terminus of a peptide or a lysine side-chain during sequence analysis by collision-induced dissociation tandem mass spectrometry. Our data provide evidence for the first case of a prokaryotic carbamylated electron transport protein occurring in vivo.     

Chiral quantification of D-, L-phenylglycine mixture using mass spectrometric kinetic method

A novel mass spectrometric method is applied to rapid, accurate, quantitative analysis of chiral phenylglycine. Transition-metal-bound complex ions containing the chiral phenylglycine are generated by electrospray ionization mass spectrometry and subjected to collision-induced dissociation. The ratio of the two competitive dissociation rates is related to the enantiomeric composition of the mixture, allowing the determination of enantiomeric contamination in the intermediates.     

Detailed elucidation of hydrocarbon contamination in food products by using solid-phase extraction and comprehensive gas chromatography with dual detection

The present work is focused on the development/optimization of a comprehensive two-dimensional gas chromatography method, with dual detection [flame ionization (FID) and mass spectrometric], for the simultaneous identification and quantification of mineral-oil contaminants in a variety of food products. The two main classes of contaminants, namely saturated and aromatic hydrocarbons, were previously fractionated on a manually-packed silver silica solid-phase extraction (SPE) cartridge. The quantitative results were compared with those obtained by performing a large volume injection, in a GC-FID system, after the same SPE process and by an on-line liquid–gas chromatography method, with very similar results observed. The presence of a series of unknown compounds, that appeared when using the off-line methods, was investigated using the mass spectrometric data, and were tentatively-identified as esterified fatty acids, most probably derived from vegetable oil based ink.     

Identification and characterization by high-performance liquid chromatography/electrospray ionization mass spectrometry of a new variant hemoglobin, Mataro [beta134(H12) Val > Ala

This work illustrates the practical use of combined microbore reversed-phase high-performance liquid chromatography (RP-HPLC) with electrospray ionization mass spectrometry (ESI-MS) in protein identification. The approach consisted of the detection of the abnormal beta-globin chain by ESI-MS analysis of mixtures of intact globins, which simultaneously provided their molecular masses. Separation of the polypeptide globin chains was carried out using microbore C4 RP-HPLC on-line with ESI-MS. Direct peptide-mapping ESI-MS without previous chromatographic separation was performed in order to identify tryptic peptides from whole blood. For the sequence confirmation of the abnormal peptide containing the mutation point, C18 RP-HPLC tryptic separation of the globin mixture on-line with collision-induced dissociation (CID) fragmentation was done. The y series ions allowed the identification of the hemoglobin (Hb) variant as [beta134(H12) Val > Ala]. This new Hb was named Hb Mataró, after the city where it was detected.