Capillary liquid chromatography/atmospheric-pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry: a comparison with liquid chromatography/matrix-assisted laser desorption/ionisation time-of-flight and liquid chromatography/electrospray ionisation quadrupole time-of-flight for the identification of tryptic peptides

The atmospheric-pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI-QIT) analysis of tryptic peptides is reported following capillary liquid chromatographic (LC) separation and direct analysis of a protein digest. Peptide fragments were identified by peptide mass fingerprinting from mass spectrometric data and sequence analysis obtained by tandem mass spectrometry of the principal mass spectral peaks using a data-dependent scanning protocol. These data were compared with those from mass spectrometric analysis using capillary LC/MALDI-time-of-flight (TOF) and capillary LC/electrospray ionisation (ESI)-quadrupole TOF. For all three configurations the resulting data were searched against the MSDB database, using MASCOT and the sequence coverage compared for each technique. Complementary data were obtained using the three techniques.     

Characterizing DNA photo-oxidation reactions by high-resolution mass measurements with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

High-resolution mass measurements by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were employed to characterize laser-induced oxidation of guanine in a small synthetic deoxyoligonucleotide. The oligonucleotide was exposed to high-intensity UV radiation at 266 nm to produce modifications on the guanine base. The primary product showed a +16 Da mass shift relative to the original strand, whereas secondary products showed mass shifts of +32 and +34 Da. The mass shift of the primary product is consistent with an 8-oxoguanine modification. However, the reactivity of the primary product with hot piperidine and other secondary oxidizing agents was different from that of a synthetic oligonucleotide containing 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxoG). Based upon the results, a new reaction scheme involving the formation of an epoxide ring across the C-4 and C-5 positions by UV laser-induced oxidation is suggested. The results also illustrate the ability of MALDI to characterize chemical reactivity rapidly at the a low picomolar level.     

Identification of degradation products formed during performic oxidation of peptides and proteins by high-performance liquid chromatography with matrix-assisted laser desorption/ionization and tandem mass spectrometry

Oxidation of proteins with performic acid is extensively used to cleave disulfide bonds. Due to its efficiency and many other advantages it deserves more attention especially in proteomics as a method for sample treatment. However, some unwanted degradations can occur during performic oxidation. In this work the degradation products during performic oxidation of two peptides and bovine serum albumin as model substrates were explored by coupling high-performance liquid chromatography (HPLC) to matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-TOF/TOFMS). In addition to well-known modifications such as oxidation of tryptophan and oxidation and chlorination of tyrosine, novel degradation products including nonspecific cleavage after asparagine or tryptophan, formylation of lysine, and beta-elimination of cysteine, were observed. Although almost all of these modification/degradation products except oxidation products of tryptophan were formed at sub-stoichiometric levels, they can cause confusion as a result of the sensitivity of mass spectrometry in analysis of the oxidized samples, especially in proteomics research. The results presented here will facilitate the interpretation of analytical data for performate-oxidized samples, and help to select appropriate methods for each unique sample.     

Identification of unusual (modified and non-encoded) amino acid residues in peptides by combinations of high-performance liquid chromatography and high-performance capillary electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The techniques for micro-level analysis of some widespread unusual amino acids (phosphorylated and hydroxylated ones) as well as of some genetically non-encoded amino acids were developed for their subsequent identification in the peptide and protein amino acid sequence by narrow-bore column high-performance liquid chromatography (10 pmol of the sample), high-performance capillary electrophoresis (1–10 pmol), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (1–10 pmol), and automatic protein gas phase sequencing (1–50 pmol).     

Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry

A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins.     

Characterization of peptides from Aplysia using microbore liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry guided purification

Liquid chromatography (LC) has been used extensively for the separation and isolation of peptides due to its high selectivity and peak capacity. An approach combining microbore LC with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) detection is described to identify peptides in cells and guide the purification of peptides from the marine mollusc Aplysia californica. Direct MALDI-MS of neurons and processes provides molecular mass information for unknown peptides with almost no sample preparation, and LC-MALDI-MS allows the isolation and purification of these peptides from pooled samples, thus enabling new putative neuropeptides to be isolated from complex cellular samples. Both direct MALDI-MS and LC-MALDI-MS are compared in terms of detecting peptides from neuronal samples. Using both approaches, two peaks from Aplysia californica connectives having molecular masses of 5013 and 5021 have been isolated, partially sequenced and identified as novel collagen-like peptides.     

Determination of disulfide bond patterns in laminin beta1 chain N-terminal domains by nano-high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry

The disulfide bonding patterns in the N-terminal (LN) domains of the basement membrane protein laminin beta1 have not been investigated so far. We report an in-depth mass spectrometric analysis using offline nano-high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (nano-HPLC/MALDI-TOF/TOF-MS) for determining the disulfide bond patterns in the LN-domain of recombinant mouse laminin beta1 chain for the first time. Mass spectra were recorded and the putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the disulfide bond. Screening the fragment ion mass spectra of disulfide-linked peptides for characteristic 66-amu patterns (34 u +32 u), arising from symmetric and asymmetric cleavage of disulfide bonds, facilitated their identification. Using various enzymes for proteolytic digestion of a recombinant laminin beta1 chain N-terminal protein fragment, a linear bonding pattern of the eight cysteine residues in the LN-domain of the laminin beta1 chain was observed with a (1-2, 3-4, 5-6, 7-8) connectivity of cysteines. The identical disulfide-bonding pattern was found in E4, the N-terminal laminin beta1 chain fragment derived by elastase digestion of mouse tumor laminin-111, confirming that this pattern also occurs in native laminin.     

Amino acid and peptide conjugates of protoporphyrin: preparation and analysis by high-performance liquid chromatography,high-performance liquid chromatography/electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Amino acid and peptide conjugates of protoporphyrin have been prepared by reacting protoporphyringen with cysteine, glutathione and peptides containing a free thiol group under acidic conditions. The conjugates were formed by the addition of the thioamino acids or peptides to the vinyl groups of protoporphyrin during the autoxidation of protoporphyinogen to protoporphyrin and is free-radical-mediated. The conjugates were separated by high-performance liquid chromatography (HPLC) and characterized by HPLC/electrospray ionization mass spectrometry (HPLC/ESI-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). All the conjugates formed were diconjugates consisting of diastereoisomers.     

Ganglioside analysis by thin-layer chromatography matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry

Metastable decomposition of ions generated in matrix-assisted laser desorption/ionization (MALDI) mass spectrometers complicates analysis of biological samples that have labile bonds. Recently, several academic laboratories and manufacturers of commercial instruments have designed instruments that introduce a cooling gas into the ion source during the MALDI event and have shown that the resulting vibrational cooling stabilizes these labile bonds. In this study, we compared stabilization and detection of desorbed gangliosides on a commercial orthogonal time-of-flight (oTOF) instrument with results we reported previously that had been obtained on a home-built Fourier transform mass spectrometer. Decoupling of the desorption/ionization from the detection steps resulted in an opportunity for desorbing thin-layer chromatography (TLC)-separated gangliosides directly from a TLC plate without compromising mass spectral accuracy and resolution of the ganglioside analysis, thus coupling TLC and oTOF mass spectrometry. The application of a declustering potential allowed control of the matrix cluster and matrix adduct formation, and, thus, enhanced the detection of the gangliosides.     

Tyrosinase-induced cross-linking of tyrosine-containing peptides investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Tyrosinase-induced oxidation of tyrosine is known to lead to melanin by cross-linking of 5,6-dihydroxyindole (DHI) and indole-5,6-quinone intermediates. However, tyrosinase-induced cross-linking of tyrosine-containing peptides has not been reported. We observed tyrosinase-induced adducts of tyrosine-containing peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). MALDI-TOFMS was also used to observe tyrosine adducts at various levels of oxidation derived from acid hydrolysis of the peptide adducts. The rate of tyrosinase-induced browning of lys-tyr-lys was about half of that of tyrosine. These results indicate that tyrosinase-induced browning of tyrosine-containing peptides via direct oxidation and cross-linking of the benzene ring of the tyrosine residue occurs at a significant rate and needs to be considered in melanogenesis.     

Characterization of silsesquioxanes by size-exclusion chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Liquid chromatography in combination with spectroscopic methods like matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) or nuclear magnetic resonance (NMR) spectroscopy is a powerful method to characterize silsesquioxanes and silsesquioxane mixtures. As new examples, the formation of silsesquioxyl-substituted silsesquioxanes [(n-octyl)(7)(SiO(1.5))(8)](2)O and [(n-octyl)(7)(SiO(1.5))(8)O](2)[(n-octyl)(6)(SiO(1.5))(8)] as well as the cage rearrangement of octa-[(n-heptyl)silsesquioxane] to larger structures [(n-heptyl)SiO(1.5))](n) up to n=28 are shown.     

Analysis of brain lipids by directly coupled matrix-assisted laser desorption ionization time-of-flight mass spectrometry and high-performance thin-layer chromatography

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a soft ionization MS technique providing only minor fragmentation of the analyte. Therefore, the method is basically suitable for mixture analysis, although the ion yields strongly depend on the basicity/acidity of the analyte in relation to the applied matrix. Accordingly, less sensitively detectable compounds may be suppressed by more sensitively detectable compounds. Thus, separation of the mixture into the individual compounds is normally indispensable. This paper demonstrates the capabilities and limitations of a direct, simple, and inexpensive MALDI-high-performance thin-layer chromatography (HPTLC) coupling for the analysis of a crude lipid extract from porcine brain. Brain lipids were chosen because they represent a rather complex mixture and are of currently significant research interest. It was found that normal-phase HPTLC-separated lipids can be easily characterized by direct MALDI-TOF-MS analysis with sufficient resolution to allow the assignment of virtually all lipid classes, even rather minor species such as phosphorylated phosphoinositides or complex glycolipids as gangliosides. Advantages and disadvantages of this approach are discussed.     

In-source decay characteristics of peptides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In-source decay (ISD) of peptides, coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, has been examined to determine the influence of the matrix, the susceptibility of amino-acid residues to ISD, and the effect of extraction delay times. Out of nine di- and tri-hydroxybenzoic acids and three cinnamic derivatives tested, the most suitable matrix for ISD was 2,5-dihydroxybenzoic acid. The amine bond at Xxx-Gly and Xxx-Val residues was less susceptible than other amino-acid residues to ISD; however, the more sensitive residue(s) were not as clear. Using a peptide that gave the y(n)- and (z(n) + 2)-series product ions, it was confirmed that amide-bond cleavage (formation of the y(n)-series ions) accompanied metastable peaks, whereas metastable peaks were never observed with amine-bond cleavage [formation of the (z(n) + 2)-series ions]. Furthermore, abundant c(n)-series ions, which originate from amine-bond cleavage on the peptide backbone, were observed whenever a minimum delay time of 38 ns or continuous extraction was used to obtain spectra. These data indicate that amine-bond cleavage in ISD takes place on the ionization time scale before the energy randomization is completed.     

Hyphenation of capillary high-performance liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for nano-scale screening of single-bead combinatorial libraries

This paper focuses on the technical aspects of chemical screening from 384-well plate nano-scale single-bead combinatorial libraries. The analytical technique utilized is a combination of capillary liquid chromatography with ultraviolet detection and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The HPLC/MALDI-MS hyphenation is achieved by means of a micro-fraction collector with a peak detection system that automatically collects the peaks onto the MALDI targets for subsequent characterization. Several experimental parameters such as type of 384-well plate, well-plate sealing foils, and a column-switching procedure were investigated using a small test library of nine components. Additionally, the influence of different MALDI matrices, different MALDI targets and sample-spotting techniques on the MALDI detection sensitivity as well as the ruggedness and sample throughput capacity of this technique were studied. Optimum results for the analytes investigated were obtained with 2,5-dihydroxybenzoic acid using on-line mixing of HPLC effluent and matrix solution. To demonstrate the potential of this capillary HPLC/MALDI-TOFMS method, its application to several single-bead libraries was investigated. The instrumental method allowed for the rapid identification and purity assessment of combinatorial libraries with detection limits down to the higher femtomole level using both UV detection and MALDI mass spectrometry.     

Proteomic analysis of rat plasma by two-dimensional liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers therefore requires either the specific depletion of high abundance proteins using immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe a two-dimensional (2D) liquid chromatography separation method for the fractionation of rat plasma. In the first dimension proteins were separated by chromatofocusing according to their isoelectric point (pI). In the second dimension, proteins were further fractionated by non-porous, reversed-phase chromatography according to their hydrophobicity. The data from both separations was displayed as a 2D protein expression map of pI versus retention time (relative hydrophobicity). Both separations were carried out on the ProteomeLab PF 2D system (Beckman Coulter), an instrument platform that provides a high degree of automation and real-time monitoring of the separation process. The reproducibility of the first-dimension separation was evaluated in terms of pH gradient formation. The second-dimension separation was evaluated in terms of peak retention times on the reversed-phase column. We found in four consecutive chromatofocusing separations that the pH gradient differed by less than 0.2 pH units at any time during the elution step. Second dimension retention times of peaks from identical pI fractions differed by less than 7 s in six consecutive separations. Each 2D separation generated a total of 540 fractions which were analyzed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). We detected approximately 275 peptides and proteins with molecular masses ranging from 3 to 225 kDa. Most fractions were found to contain multiple low and high molecular weight proteins. Differential display of 2D protein expression maps from retinol-sufficient and -deficient rat plasma samples identified a fraction with several proteins that appeared to be down-regulated in the vitamin A-deficient animal. Quantitative proteomic analysis of complex samples such as plasma is still a difficult task. We discuss the potential of this approach for biomarker discovery and address the experimental challenges that remain.     

A high-performance matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometer with collisional cooling

A high-performance orthogonal time-of-flight (TOF) mass spectrometer was developed specifically for use in combination with a matrix-assisted laser desorption/ionization (MALDI) source. The MALDI source features an ionization region containing a buffer gas with variable pressure. The source is interfaced to the TOF section via a collisional focusing ion guide. The pressure in the source influences the rate of cooling and allows control of ion fragmentation. The instrument provides uniform resolution up to 18,000 FWHM (full width at half maximum). Mass accuracy routinely achieved with a single-point internal recalibration is below 2 ppm for protein digest samples. The instrument is also capable of recording spectra of samples containing compounds with a broad range of masses while using one set of experimental conditions and without compromising resolution or mass accuracy.     

Improved mass accuracy in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides

One problem of matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometry is the moderate mass accuracy that typically can be obtained in routine applications, Here we report improved mass accuracy for peptides, even when low amounts and complex peptide mixtures are used. A new procedure for preparing matrix surfaces is used, and there is no need to mix the matrix with the sample or to add internal standards. Examples are shown with a mass accuracy better than 50 ppm in a peptide mixture. Peptide mapping as well as sequencing by creating “ragged ends” or “ladder sequencing” should benefit especially from the improved mass accuracy.     

Analysis of bacterial lipodepsipeptides by matrix-assisted laser desorption/ionisation time-of-flight and high-performance liquid chromatography with electrospray mass spectrometry

Strains of certain plant pathogenic bacteria, in particular several pathovars of Pseudomonas syringae, are known to produce cyclic lipodepsipeptides (LDPs) endowed with peculiar structural features and noticeable biological activities. In this study, a mass spectrometry procedure is proposed for screening LDP-producing bacterial strains and for identifying and assessing individual LDPs. After matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) screening of thirteen P. syringae strains for LDP production, the extracts from culture filtrates of eight positive strains were subjected to electrospray mass spectrometry for the identification of LDPs. Five strains were found to produce two forms of syringomycins (SR-E and SR-G) and two forms of syringopeptin 25 (SP25A and SP25B); two strains produced SR-E, SR-G and a new form of SP22; one strain produced syringotoxin (ST) and syringostatin A (SS-A) in addition to SP25A and SP25B. The yield in culture of two major LPDs: SR-G (3.2-13.8 mg x L(-1)) and SP25A (41.6-231.5 mg x L(-1)) was assessed by and high-performance liquid chromatography with electrospray mass spectrometry (HPLC/ESI-MS) in both scan and single ion monitoring (SIM) modes. Results of this investigation showed that the mass spectrometry protocol developed here is a precise and reliable method for screening bacterial strains for LDP production and for assessing the amount of each metabolite under various culture conditions. This could be of practical value in view of potential applications, e.g. biocontrol of post-harvest fungal diseases.     

Characterization of mesogen-jacketed liquid crystalline polymers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

For synthetic polymers, a proper sample preparation method is essential for successful characterization by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In this work, six synthetic mesogen-jacketed liquid crystalline polymers (MJLCPs) with different main-chain, spacer and mesogenic units were investigated by MALDI-TOF mass spectrometry. Several factors that affect the analysis of these polymers were examined. These factors include matrices used, cationization salts used, the concentration of polymers, and the ratio of sample to matrix. After testing different conditions, we found a suitable sample preparation method for these six polymers. The number average molecular weight (M(n)), weight average molecular weight (M(w)) and polydispersity (PD) were calculated using data obtained in the linear mode. The end groups of the polymers were proposed using data obtained in reflectron mode. Copyright 2000 John Wiley & Sons, Ltd.