Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to rapidly screen for albuminuria

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is used as an alternative method for the rapid diagnosis of albuminuria. This technique requires no further sample pretreatment than simply mixing the urine sample with a MALDI matrix and drying under ambient conditions. The resulting MALDI mass spectra reveal albumin ions having charges ranging from +1 to +5. The detection of albumin is possible using any of the three most common MALDI matrices - sinapinic acid (SA), 2,5-dihydroxybenzoic acid (2,5-DHB), or 4-hydroxy-alpha-cyanocinnamic acid (alpha-CHC). Using this analytical approach, the limit of detection for albumin in urine is 10(-6) M, approximately 5 to 10 times lower than that detectable through conventional chemical testing.     

Intact protein analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Direct tandem mass spectrometric (MS/MS) analysis of small, singly charged protein ions by tandem time-of-flight mass spectrometry (TOFMS) is demonstrated for proteins up to a molecular mass of 12 kDa. The MALDI-generated singly charged precursor ions predominantly yield product ions resulting from metastable fragmentation at aspartyl and prolyl residues. Additional series of C-terminal sequence ions provide in some cases sufficient information for protein identification. The amount of sample required to obtain good quality spectra is in the high femtomolar to low picomolar range. Within this range, MALDI-MS/MS using TOF/TOF trade mark ion optics now provides the opportunity for direct protein identification and partial characterization without prior enzymatic hydrolysis.     

Decomposition of protein nitrosothiolsin matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The S-nitrosylation of proteins is involved in the trafficking of nitric oxide (NO) in intra- and extracellular milieus. To establish a mass spectrometric method for identifying this post-translational modification of proteins, a synthetic peptide and transthyretin were S-nitrosylated in vitro and analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The intact molecular ion species of nitrosylated compounds was identified in the ESI mass spectrum without elimination of the NO group. However, the labile nature of the S-NO bond was evident when the in-source fragmentation efficiently generated [M + H - 30](+) ions. The decomposition was prominent for multiply charged transthyretin ions with high charge states under ordinary ESI conditions, indicating that the application of minimum nozzle potentials was essential for delineating the stoichiometry of nitrosylation in proteins. With MALDI, the S-NO bond cleavage occurred during the ionization process, and the subsequent reduction generated [M + H - 29](+) ions.     

In-situ monitoring of protein labeling reactions by matrix-assisted laser desorption/ionization mass spectrometry

Taking the labeling reaction of horse heart cytochrome c or ubiquitin with biotinamidocaproate N-hydroxysucchinimide ester (biotin-NHS) as test cases, this report demonstrates the usefulness of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for in-situ monitoring of the labeling process and for determining the composition of the labeled products without the need for prior separation. The effects of pH and starting materials concentration on the labeling process were investigated in detail. Our MALDI MS results show that: (1) labeled products are always mixtures of different conjugates, which may explain peak broadening found in chromatographic studies of labeling reactions; (2) the higher conjugate fractions become more prominent as the labeling reaction proceeds, with a concomitant exponential decline of the lower conjugate fractions; (3) biotin-NHS can be incorporated into peptides and protein in a stepwise and controlled manner simply by adjusting the molar ratio of the starting materials.     

In-situ monitoring of protein labeling reactions by matrix-assisted laser desorption/ionization mass spectrometry

Taking the labeling reaction of horse heart cytochrome c or ubiquitin with biotinamidocaproate N-hydroxysucchinimide ester (biotin-NHS) as test cases, this report demonstrates the usefulness of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for in-situ monitoring of the labeling process and for determining the composition of the labeled products without the need for prior separation. The effects of pH and starting materials concentration on the labeling process were investigated in detail. Our MALDI MS results show that: (1) labeled products are always mixtures of different conjugates, which may explain peak broadening found in chromatographic studies of labeling reactions; (2) the higher conjugate fractions become more prominent as the labeling reaction proceeds, with a concomitant exponential decline of the lower conjugate fractions; (3) biotin-NHS can be incorporated into peptides and protein in a stepwise and controlled manner simply by adjusting the molar ratio of the starting materials.     

Miniaturizing sample spots for matrix-assisted laser desorption/ionization mass spectrometry

The trend of miniaturization in bioanalytical chemistry is shifting from technical development to practical application. In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), progress in miniaturizing sample spots has been driven by the needs to increase sensitivity and speed, to interface with other analytical microtechnologies, and to develop miniaturized instrumentation.We review recent developments in miniaturizing sample spots for MALDI-MS. We cover both target modification and microdispensing technologies, and we emphasize the benefits with respect to sensitivity, throughput and automation.We hope that this review will encourage further method development and application of miniaturized sample spots for MALDI-MS, so as to expand applications in analytical chemistry, protein science and molecular biology.     

On-line atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

A new technique is presented for the coupling of atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) mass spectrometry with liquid delivery systems. Mass measurements of polymers and peptides are demonstrated using a co-dissolved matrix, e.g. alpha-cyano-4-hydroxycinnamic acid (HCCA). Improvements in terms of sensitivity are achieved by optimizing the shape und control of the exit capillary and by using a laser (355 nm) at a 1 kHz repetition rate. Two calibration experiments promise a good applicability of the presented coupling method for quantitative measurements. The limit of detection achieved so far is 500 nM for peptides in methanol solution containing 25 mM HCCA.     

Improved method for peak picking in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method for peak picking for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described. The method is based on the assumption that two sets of ions are formed during the ionization stage, which have Gaussian distributions but different velocity profiles. This gives rise to a certain degree of peak skewness. Our algorithm deconvolutes the peak and utilizes the fast velocity, bulk ion distribution for peak picking. Evaluation of the performance of the new method was conducted using peptide peaks from a bovine serum albumin (BSA) digest, and compared with the commercial peak-picking algorithms Centroid and SNAP. When using the new two-Gaussian algorithm, for strong signals the mass accuracy was equal to or marginally better than the results obtained from the commercial algorithms. However, for weak, distorted peaks, considerable improvement in both mass accuracy and precision was obtained. This improvement should be particularly useful in proteomics, where a lack of signal strength is often encountered when dealing with weakly expressed proteins. Finally, since the new peak-picking method uses information from the entire signal, no adjustments of parameters related to peak height have to be made, which simplifies its practical use.     

Detection of immune complexes by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to detect an immune complex formed between beta-lactoglobulin and polyclonal anti-beta-lactoglobulin antibody in the gas phase. The most important experimental parameters to detect such a specific antibody-antigen complex by MALDI were the use of solutions at near-neutral pH and of sinapinic acid matrix prepared by the dried-droplet method. Under such conditions, predominantly one but also two molecules of antigen protein were complexed by the antibody. Specific formation of the antibody-antigen complex was confirmed by performing competitive reactions. Addition of antibody to a 1:1 mixture of beta-lactoglobulin and one control protein resulted not only in the appearance of the expected antibody-antigen complex, but also in a strong decrease in the free beta-lactoglobulin signal, while the abundance of the control protein was not influenced.     

Detergent enhancement of on-tissue protein analysis by matrix-assisted laser desorption/ionization imaging mass spectrometry

Matrix-Assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) is a molecular technology that allows simultaneous investigation of the content and spatial distribution of molecules within tissue. In this work, we examine different classes of detergents, the anionic sodium dodecyl sulfate (SDS), the nonionic detergents Triton X-100, Tween 20 and Tween 80, and the zwitterionic 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) for use in MALDI IMS of analytes above m/z 4000. These detergents were found to be compatible with MALDI MS and did not cause signal suppression relative to non-detergent applications and did not produce interfering background signals. In general, these detergents enhanced signal acquisition within the mass range m/z 4-40 000. Adding detergents into the matrix was comparable with the separate application of detergent and matrix. Evaluation of spectra collected from organ-specific regions of a whole mouse pup section showed that different detergents perform optimally with different organs, indicating that detergent selection should be optimized on the specific tissue for maximum gain. These data show the utility of detergents towards enhancement of protein signals for on-tissue MALDI IMS analysis.     

Serum protein profiling by miniaturized solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

Serum profiling by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) holds promise as a clinical tool for early diagnosis of cancer and other human diseases. Sample preparation is key to achieving reproducible and well-resolved signals in MALDI-MS; a prerequisite for translation of MALDI-MS based diagnostic methods to clinical applications. We have investigated a number of MALDI matrices and several miniaturized solid-phase extraction (SPE) methods for serum protein concentration and desalting with the aim of generating reproducible, high-quality protein profiles by MALDI-MS. We developed a simple protocol for serum profiling that combines a matrix mixture of 2,5-dihydroxybenzoic acid and alpha-cyano-4-hydroxycinnamic acid with miniaturized SPE and MALDI-MS. Functionalized membrane discs with hydrophobic, ion-exchange or chelating properties allowed reproducible MALDI mass spectra (m/z 1000-12,000) to be obtained from serum. In a proof-of-principle application, SPE with chelating material and MALDI-MS identified protein peaks in serum that had been previously reported for distinguishing a person diagnosed with breast cancer from a control. These preliminary results indicate that this simple SPE/MALDI-MS method for serum profiling provides a versatile and scalable platform for clinical proteomics.     

Improved analysis of membrane protein by PVDF-aided, matrix-assisted laser desorption/ionization mass spectrometry

Characterization of membrane proteins remains an analytical challenge because of difficulties associated with tedious isolation and purification. This study presents the utility of the polyvinylidene difluoride (PVDF) membrane for direct sub-proteome profiling and membrane protein characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The hydrophobic adsorption of protein, particularly membrane proteins, on the PVDF surface enables efficient on-PVDF washing to remove high concentrations of detergents and salts, such as up to 5% sodium dodecyl sulfate (SDS). The enhanced spectrum quality for MALDI detection is particularly notable for high molecular weight proteins. By using on-PVDF washing prior to MALDI detection, we obtained protein profiles of the detergent-containing and detergent-insoluble membrane fractions from Methylococcus capsulatus (Bath). Similar improvements of signal-to-noise ratios were shown on the MALDI spectra for proteins electroblotted from SDS-polyacrylamide gel electrophoresis (SDS-PAGE) onto the PVDF membrane. We have applied this strategy to obtain intact molecular weights of the particulate methane monooxygenase (pMMO) composed of three intrinsic membrane-bound proteins, PmoA, PmoB, and PmoC. Together with peptide sequencing by tandem mass spectrometry, post-translational modifications including N-terminal acetylation of PmoA and PmoC and alternative C-terminal truncation of PmoB were identified. The above results show that PVDF-aided MALDI-MS can be an effective approach for profiling and characterization of membrane proteins.     

Differentiation of enantiomers using matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has successfully been used to differentiate pseudo-enantiomeric (isotopically labelled) amino acids by using cyclodextrin as complexing host. By using different pseudo-enantiomeric mixtures (i.e. R(Dn) + S; and R + S(Dn)), it has been demonstrated that the preference of cyclodextrin for S-enantiomers is not due to the size differences caused by the hydrogen/deuterium substitution. It is postulated that this method can be extended to differentiate enantiomers (and determine enantiomeric excess) by using a pair of enantiomeric hosts, as demonstrated previously using other ionization techniques, but with much higher sensitivity.     

Rapidly switchable matrix-assisted laser desorption/ionization and electrospray quadrupole-time-of-flight mass spectrometry for protein identification

We describe a new interface for a prototype quadrupole-quadrupole-time-of-flight (TOF) mass spectrometer (Centaur, Sciex) that allows rapid switching between electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) modes of operation. Instrument performance in both modes is comparable (i.e., resolution approximately 10,000 FWHM, mass accuracy <10 ppm, sensitivity approximately 1 fmol) because the ion source is decoupled from the TOF mass analyzer by extensive gas collisions in the quadrupole stages of the instrument. The capacity to obtain side-by-side high quality ESI and MALDI mass spectra from a single proteolytic mixture greatly facilitates the identification of proteins and elucidation of their primary structures. Improved strategies for protein identification result from this ability to measure spectra using both ionization modes in the same instrument and to perform MS/MS on singly charged as well as multiply charged ions. Examples are provided to demonstrate the utility and performance of the modified instrument.     

Covariance mapping in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A novel method for acquisition and numerical analysis of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectral data is described. The digitized ion current transient from each consecutive laser shot is first acquired and stored independently. Subsequently, statistical correlation parameters between all stored transients are computed. We illustrate the uses of this event-by-event analysis method for studies of sample surface heterogeneity as well as for elucidating the mechanisms of ion formation in MALDI. Other potential applications of the method are also outlined.     

Analysis of solid-supported oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This study presents matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) as a powerful tool to analyze and characterize oligonucleotides covalently linked to a solid support during their synthesis. The analysis of the fragment ions generated either in negative or positive mode allows direct and easy access to the nucleotide sequence and identification of the internucleosidic linkage. The mechanisms of the fragmentation of the solid-supported oligonucleotides induced by MALDI-TOFMS are discussed. Copyright 2000 John Wiley & Sons, Ltd.     

Analysis of plant phosphatidylcholines by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) for the quantitative determination of phospholipid (PL) molecular species has been problematic, due primarily to the formation of multiple signals (corresponding to the molecular ion and other adducts) for some classes of PL. For example, analysis of phosphatidylcholine (PC) yielded signals that corresponded to protonated and sodiated molecules in the MALDI spectrum. The resulting spectral overlap among various molecular species (e.g. [PC(16:0/18:2) + Na] and [PC(18:2/18:3)]) made it impossible to ascertain their relative amounts using this technique. Other spectral ambiguities existed among different structural isomers, such as PC(18:1/18:1) and PC(18:0/18:2). We determined that molecular species could be resolved by MALDI-TOFMS by first removing the polar head (e.g. phosphocholine) from the phospholipid to effect production of only the sodiated molecules of the corresponding diacylglycerols (DAGs). Analysis of the resulting spectrum allowed unequivocal determination of the molecular species profile of PC from potato tuber and soybean. Estimation of fatty acid composition based on the molecular species determined by MALDI-TOFMS analysis agreed with that from GC-FID analysis. Post-source decay (PSD) was used to resolve standard isomers of PC (e.g. 18:1/18:1 vs. 18:0/18:2). Our results indicated that PSD is a useful approach for resolving structural isomers of PL molecular species.     

Sequencing of oligosaccharides by collision-induced dissociation matrix-assisted laser desorption/ionization mass spectrometry

A study of the collision-induced dissociation post-source decay (PSD) spectra of free oligosaccharides is presented. These spectra, when obtained with helium as collision gas, show (1,5)X fragments containing the reducing end sugar. The presence of these fragments permits Y ions and, consequently, B and C peaks to be identified. This is a common behaviour from which it has been possible to delineate a general method for the easy assignment of the peaks in PSD spectra of underivatized neutral sugars, allowing the sequence of a real unknown to be obtained.     

Characterization of humic substances by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and laser desorption/ionization (LDI-)TOFMS have been used to characterize Suwannee River humic substances, obtained from the International Humic Substances Society (IHSS), and Armadale soil fulvic acid (ASFA). An array of MALDI matrices were tested for use with humic substances, including alpha-cyano-4-hydroxycinammic acid (CHCA), 2-(4-hydroxyphenylazo)benzoic acid (HABA), 2,5-dihydroxybenzoic acid (DHBA), sinapinic acid, dithranol and norharmane. DHBA yielded the best results, exhibiting superior ionization efficiency, low noise, broad applicability to the analytes of interest, and most importantly producing an abundance of high mass ions, the highest observed being m/z 1848. A number of sample preparation modes were investigated; the overlayer method improved sample/matrix homogeneity and hence shot-to-shot reproducibility. The choice of the matrix, mass ratio of analyte to matrix, and the sample preparation protocol, were found to be the most critical factors governing the quality of the mass spectra. Matrix suppression was greatly enhanced by ensuring good mixing of matrix and analyte in the solid phase, proper optimization of the matrix/analyte ratio, and optimizing delayed extraction to ensure complete matrix-analyte reaction in the plume before ions are moved to the flight tube. A number of common features, in particular specific ions which could not be attributed to the matrices or to contaminants, were present in the spectra of all the humic substances, regardless of origin or operational definition. Additionally, a prominent repeating pattern of peaks separated by 55, 114 and 169 Da was clearly observed in both LDI and MALDI, suggesting that the humic compounds studied here may have quasi-polymeric or oligomeric features.     

Characterization of metal-labelled peptides by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry

Metal labelling of peptides and proteins using high-affinity metal-chelating compounds has found widespread applications in the medical and bioanalytical fields. In the present study we investigated the analysis of peptides derivatized either with cysteine- or amino group-directed metal-bound DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The metal complexes of DOTA were shown to be stable under MALDI-MS conditions. The introduction of the metal label led in a number of cases to significantly increased signal-to-noise (S/N) values and thus improved sensitivity of the labelled peptides compared to their unlabelled counterparts, especially for multiply labelled peptides. The presence of the labels did alter the tandem mass spectrometric (MS/MS) behaviour, namely the formation of sequence specific a-, b- and y-ion series, in dependence of the position of the label within the peptide sequence. For cysteine-derivatized peptides several label-specific reporter ions and characteristic immonium ions could be identified. Amino-directed labelling led only to the formation of characteristic immonium ions in ε-amino groups of lysine, whereas N-terminal labelling in some cases led to the formation of a(1)- and b(1)-ions. The results clearly show that MALDI-MS is suitable for the analysis of metal-labelled peptides, which was also confirmed in liquid chromatography (LC)/MALDI-based identification of proteins in a model protein mixture labelled with Cys-reactive DOTA. Here, in comparison to a run with alkylated cysteines, more than 50% more cysteine-containing peptides were identified.