Ionic liquids as matrices in microfluidic sample deposition for high-mass matrix- assisted laser desorption/ionization mass spectrometry

Sample preparation for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) via a microfluidic deposition device using ionic liquid matrices addresses several problems of standard protocols with crystalline matrices, such as the heterogeneity of sample spots due to the co-crystallization of sample and matrix and the limited capability for high-throughput analysis. Since ionic liquid matrices do not solidify during the measurement, the resulting sample spots are homogeneous. The use of these matrices is also beneficial for automated sample preparation, since crystallization of the matrix is avoided and, thus, no clogging of the spotting device can occur. The applicability of ionic liquids to the analysis of biomolecules with high molecular weights, up to ≈ 1 MDa is shown, as well as a good sensitivity (5 fmol) for recombinant human fibronectin, a protein with a molecular weight of 226 kDa. Microfluidic sample deposition of proteins with high molecular weights will, in the future, allow parallel sample preparation for MALDI-MS and for electron microscopy.     

Electrospray sample deposition for matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry with attomole detection limits

Electrospray sample deposition was explored for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In this method, nanoliter volumes of matrix/analyte mixture were electrosprayed from a high voltage biased (1-2 kV) fused-silica capillary onto a grounded MALDI plate mounted 100-500 microm from the capillary outlet. Electrospray deposition with these conditions produced sample spots 200-300 microm in diameter thus matching the laser spot size. Varying spray voltage and distance resulted in different crystal sizes and volatilization rates for alpha-cyano-4-hydroxycinnamic acid matrix. Best results were obtained when the sample was deposited as wet droplets as opposed to deposition as dried solid. Under 'wet-spray' conditions, 2-4 microm diameter crystals were formed and detection limits for several neuropeptides were 0.7-25 amol. Samples could be pre-concentrated on the plate by spraying continuously and allowing sample to evaporate in a small spot. Sample volumes as large as 580 nL were deposited yielding a detection limit of 35 pM for neurotensin 1-11. Electrospray sample deposition yielded similar results when using atmospheric pressure-MALDI coupled with a quadrupole ion trap mass spectrometer, except that the sensitivity was approximately seven-fold worse.     

Ready-made matrix-assisted laser desorption/ionization target plates coated with thin matrix layer for automated sample deposition in high-density array format.

The methodology for ready-made matrix-assisted laser desorption/ionization (MALDI) target plates covered with an optimized thin layer consisting of matrix and nitrocellulose has been developed. Piezoelectric microdispensing enabled sample depositions in a high-density array format of 2000 sample depositions on a conventionally sized target plate (45 x 47 mm). The sample depositions were made reproducibly in a fully automated mode by using an in-house developed computer-controlled piezoelectric flow-through microdispenser. Additionally, the piezoelectric technique facilitated significant analyte enrichment that increased the detection sensitivity. The MS signal was obtained rapidly, generally within ten laser pulses. An airbrush device was used to generate a fine spray of matrix and nitrocellulose dissolved in acetone. The acetone evaporated instantly when reaching the target plate leaving the entire surface with a thin and uniform matrix/nitrocellulose coating consisting of very small crystals of matrix embedded in the nitrocellulose. These crystals acted as a seed-layer on subsequent analyte depositions, rendering homogeneous sample spots when using alpha-cyano-4-hydroxycinnamic acid (CHCA) as matrix. The relative standard deviation of the signal intensity between spots was (20-30)% (n = 30). The detection sensitivity was improved by restricting the sample spot diameter to 300 microm. The spot size was affected by the deposition rate and the evaporation rate of the dispensed sample volume. Mass spectra of a 25-amol peptide mixture deposition were successfully recorded.     

Coupling of nanoflow liquid chromatography to matrix-assisted laser desorption/ionization mass spectrometry: real-time liquid chromatography run mapping on a MALDI plate

The major obstacle in the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) instruments in the analysis of complex proteome samples is the lack of a direct coupling of a highly resolving separation technique with the mass spectrometer itself. To overcome this drawback, a spotting device for capillary and nanoflow liquid chromatography (LC) with a special liquid deposition principle for lowest volumes was developed. The instrument is able to perform MALDI spotting in real time in order to deposit the LC run on the MALDI plate, and therefore couples the high resolution power of nano-RP-HPLC separation directly with MALDI-MS. This work describes the development and optimization of a method for spotting with online matrix addition, and illustrates its use in the analysis of a complex proteome sample.     

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.     

Capillary electrophoresis using a surfactant-treated capillary coupled with offline matrix-assisted laser desorption ionization mass spectrometry for high efficiency and sensitivity detection of proteins.

A method of combining capillary electrophoresis (CE) using a surfactant-modified capillary with matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is described for protein analysis. The CE-MALDI-MS coupling is based on CE fraction collection of nanoliter volume samples in less than 5 microl of dilute acid. This offline coupling does not require any special instrumentation and can be readily performed with commercial instruments. Protein adsorption during CE separation is prevented by coating the capillary with the surfactant didodecyldimethylammonium bromide. This surfactant binds strongly with the capillary wall, hence it does not desorb significantly to interfere with subsequent MALDI-MS analysis. It is shown that the use of a dilute acid for CE fraction collection is advantageous in lowering the detection limit of MALDI-MS compared to using an electrophoretic buffer. The detection limit for proteins such as cytochrome c is 23 fmol injected for CE, or 1.2 fmol spotted for MALDI-MS. This sensitivity is comparable to alternative CE-MALDI-MS coupling techniques using direct CE sample deposition on the MALDI target. In addition, the fraction collection approach has the advantage of allowing multiple reactions to be carried out on the fractioned sample. These reactions are very important in protein identification and structure analysis.     

Capillary isoelectric focusing hyphenated to single- and multistage matrix-assisted laser desorption/ionization-mass spectrometry using automated sheath-flow-assisted sample deposition

In this paper CIEF combined with MALDI-MS is described using a sheath-liquid-assisted automatic sample deposition from the separation capillary onto a MALDI target. Sample/matrix preparation techniques on the target resembling the dried droplet and the thin layer methods were evaluated in the context of the automatic spotting. Volatile buffers were used as IEF catholyte solutions. Test samples consisting of tryptic peptides, glycopeptides, and phosphopeptides of well-known proteins showed that CIEF-MALDI-MS can be used as effective preseparation method prior to MS, allowing to obtain the amino acid sequence coverage of proteins similar to that achieved with CZE-MALDI-MS and CZE-ESI-MS. Particularly, completeness and reliability of glycopeptide analysis is much enhanced by the preseparation. The effect is less pronounced but still significantly found with phosphopeptides present in the test protein. Finally, a test sample of five standard proteins demonstrates the suitability of this technique also for the treatment of intact proteins. This technique has potential to emerge as a faster method analogous and complementary to 2-DE and to IPG-IEF-MALDI-MS demonstrated before by the group of Loo [1].     

Electrospray interfacing of polymer microfluidics to MALDI-MS

The off-line coupling of polymer microfluidics to MALDI-MS is presented using electrospray deposition. Using polycarbonate microfluidic chips with integrated hydrophobic membrane electrospray tips, peptides and proteins are deposited onto a stainless steel target followed by MALDI-MS analysis. Microchip electrospray deposition is found to yield excellent spatial control and homogeneity of deposited peptide spots, and significantly improved MALDI-MS spectral reproducibility compared to traditional target preparation methods. A detection limit of 3.5 fmol is demonstrated for angiotensin. Furthermore, multiple electrospray tips on a single chip provide the ability to simultaneously elute parallel sample streams onto a MALDI target for high-throughput multiplexed analysis. Using a three-element electrospray tip array with 150 microm spacing, the simultaneous deposition of bradykinin, fibrinopeptide, and angiotensin is achieved with no cross talk between deposited samples. In addition, in-line proteolytic digestion of intact proteins is successfully achieved during the electrospray process by binding trypsin within the electrospray membrane, eliminating the need for on-probe digestion prior to MALDI-MS. The technology offers promise for a range of microfluidic platforms designed for high-throughput multiplexed proteomic analyses in which simultaneous on-chip separations require an effective interface to MS.     

Ionic liquid matrices with phosphoric acid as matrix additive for the facilitated analysis of phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool for the analysis and characterization of protein phosphorylation on the peptide level. In this study, the applicability of ionic liquid matrices (ILM) formed by combination of the crystalline MALDI matrix 2,5-dihydroxybenzoic acid (DHB) with pyridine or n-butylamine was tested for the analysis of phosphopeptides. Low ionization efficiency in both positive and negative ion mode was observed in acid-free sample preparations. Upon addition of 0.1% trifluoroacetic acid (TFA), ion formation was increased, but analogously to the situation described earlier for pure DHB, best results were obtained upon use of 1% phosphoric acid as matrix additive. The samples prepared in this way were significantly more homogeneous than preparations with pure DHB, thus avoiding the need for time-consuming search for hot spots. Other characteristics like metastable fragmentation of phosphopeptides did not differ from that observed in classical preparations. The limits of detection for synthetic phosphopeptides and singly or multiply phosphorylated peptides from tryptic digests of alpha- and beta-casein were comparable with those obtained when using pure DHB; in some cases even higher signal intensities could be observed in the ILM. The use of ILM in combination with 1% phosphoric acid as matrix additive significantly facilitates analysis of phosphopeptides by MALDI-MS.     

Capillary-Channeled Polymer (C-CP) Fibers as a Stationary Phase for Sample Clean-Up of Protein Solutions for Matrix-Assisted Laser/Desorption Ionization Mass Spectrometry

Capillary-channeled polymer (C-CP) fibers are employed in a micropipette tip format to affect a stationary phase for the solid phase extraction (SPE) of proteins from buffer solutions prior to MALDI-MS analysis. Proteins readily adsorb to the polypropylene (PP) C-CP fibers while buffer species are easily washed off the tips using DI-H(2)O. Elution of the solutes is achieved with an aliquot of 50:50 ACN:H(2)O, which is compatible with the subsequent spotting on the MALDI target with the matrix solution. Lysozyme and cytochrome c are used as test species, with a primary buffer composition of 100 mM Tris-HCl. In this case, direct MALDI-MS produces no discernible protein signals. SPE on the C-CP fibers yields high fidelity mass spectra for 1 μL sample volumes. Limits of detection for cytochrome c in 100 mM Tris-HCl are on the order of 40 nM. Extraction of cytochrome c from buffer concentrations of up to 1 M Tris-HCl, provides signal recoveries that are suppressed by only ~50% versus neat protein solutions. Finally, extraction of 3.1 μM cytochrome c from a synthetic urine matrix exhibits excellent recovery.     

Comparison of Novel Sampling Methods for the Analysis of Capillary Electrophoresis Fractions by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Two methods for the collection of capillary electrophoresis fractions for analysis by matrix-assisted laser desorption/ionization have been investigated; the first using a novel ion-porous joint design for dropwise fraction collection and the second, a process of electrospray deposition. Sensitivity, shot-to-shot reproducibility, effect of matrix preparation and influence of buffer additives encountered in micellar electrokinetic capillary chromatography (MECC) have been examined using a standard peptide, methionine enkephalin. The direct collection of droplets from the capillary outlet was found to give a superior signal intensity, whereas the electrospray method, in its first stage of development, suffered through dilution effects and excessively wide sample distribution. The target compound was successfully collected by electrospray deposition and results indicated that signal intensity and sample homogeneity would be enhanced if the sprayed output could be collected into the sample wells in its entirety. The advantage of using electrospray deposition for the preparation of matrix layers followed by direct droplet collection onto the electrosprayed matrix was clearly demonstrated by improvements in both sensitivity and shot-to-shot variance in signal intensity across the sample well compared with collection onto spotted matrix. In addition, compatibility of the droplet collection method with MECC was illustrated by the recovery of the compound of interest from a buffer containing 30 mm sodium dodecyl sulphate. © 1997 John Wiley & Sons, Ltd.     

Nanodiamond MALDI support for enhancing the credibility of identifying proteins

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a standard analytical tool for protein identification and peptide sequencing. High sensitivity and resolution are two critical parameters for recording good peptide mass fingerprinting (PMF) of low abundance proteins. Here, we report a novel nanodiamond (ND) (normal size 3–10 nm) support for MALDI-MS target, over which -cyano-4-hydrocinnamic acid (CCA) crystallizes evenly. Good reproducibility of relative peak intensity (R.S.D. less than 11.8%) among sample spot (from ring to center) is achieved on ND support. Therefore, the search for “hot spots” during the analysis is not necessary, which is supporting for the automatic acquisition of data. Due to high absorbability of energy from the laser, the ND support improves ionization efficiency of samples. In general, the sensitivity of MS obtained on ND support can be enhanced three to four times compared to the conventional MALDI sample preparation technique. Sensitivity obtained on ND support ranges from 62.5 amol of Arg-vasopressin standard peptide to 1.0 fmol of myoglobin tryptic peptide mixture. Reduced spot size and increased sensitivity in MALDI-MS are also accomplished by ND support. With spot size reduced, the signal intensity of cytochrome c (Cyt c) tryptic peptide obtained on ND support is at least seven times greater than it acquired on stainless steel. And ND support has been found better tolerance for salt (up to 500 mM NaCl) to MALDI-MS analysis. All these properties make ND support a valuable tool for MALDI-MS identification of proteins.     

Method for homogeneous spotting of antibodies on membranes: application to the sensitive detection of ochratoxin A

Membrane-based dot immunoassays are now widely used in almost every branch of biology and medicine. However, the quality of the immobilized antigen or antibody spots on the membranes was found to be highly operator-dependent and spotting by conventional methods often leads to heterogeneous spot morphologies and deposition inconsistencies. To circumvent these problems, a spotting method has been developed which is based on focussed absorption of an applied antibody solution through an aqueous network of capillary channels formed between the membrane and a wetted absorbent body. The method does not require any equipment for creating vacuum and according to assay requirements highly homogeneous spots of uniform size, in the range of 0.8- to 9-mm diameter, can be obtained by varying the volume of the applied antibody solution. Spot intensities were sufficiently high even at high antibody dilutions. Immobilization of anti-ochratoxin A (anti-OA) antibody by this method gave 2-fold increased sensitivity in a competitive assay of the toxin compared to conventional spotting methods. The calculated CV of the colour intensity for spots of different sizes (0.8 to 9 mm) was between 4.5 and 1%. Application of this spotting technique has been demonstrated for detection of OA in wine and coffee samples with the elimination of matrix interferences in the same immunoassay system. This was achieved by selective removal of nonspecific interfering substances from the sample extract during the assay. The detection limit of OA in wine (1 μg L⁻¹) and coffee (2.5 μg kg⁻¹) obtained by the present new method is superior to values reported recently. Thus, the present new method will be highly useful for improved performance of membrane-based immunoassays in almost every branch of biology and medicine.      

Nanoliter deposition unit for pipetting droplets of small volumes for Total Reflection X-ray Fluorescence applications

A nanoliter droplet deposition unit was developed and characterized for application of sample preparation in TXRF. The droplets produced on quartz reflectors as well as on wafers show a good reproducibility, also the accuracy of the pipetted volume could be proved by a quantitative TXRF analysis using an external standard. The samples were found to be independent of rotation of the sample carrier. Angle scans showed droplet residue behavior, and the fluorescence signal is relatively invariant of the angle of incidence below the critical angle, which is useful for producing standards for external calibration for semiconductor surface contamination measurements by TXRF. Further it could be demonstrated that the nanoliter deposition unit is perfectly able to produce patterns of samples for applications like the quantification of aerosols collected by impactors.     

Direct matrix-assisted laser desorption ionization mass spectrometric analysis of proteins immobilized on nylon-based membranes

Direct analysis of proteins adsorbed onto the surface of nylon membranes has been performed at the picomole level by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Nylon-66 and positive charge-modified nylon (Zetabind) membranes fixed to MALDI probe tips were successfully employed to analyze picomole quantities of sample that were adsorbed onto these inert supports prior to adding a matrix-containing solution. Proteins and peptides are readily solubilized from these types of membrane with conventional matrix solvents and cocrystallize with the matrix on the membrane surface. Because solubilization of membrane-adsorbed protein is necessary for successful sample preparation, nylon membranes are more suitable for use with MALDI-MS than other protein transfer membranes such as polyvinylidene difluoride or nitrocellulose. When compared to samples prepared conventionally, no apparent loss of sensitivity or resolution is observed when analysis by MALDI-MS is performed from nylon-66 or positive charge-modified nylon membranes. Detection limits and resolution are not apparently affected by the membrane immobilization/washing procedure, and no change in the mass accuracy is observed when analysis is performed on the nylon surface. However, there is a time shift (increase) in ion flight time when analysis by MALDI-time-of-flight-MS is performed directly from the membrane fixed to the probe tip (about 200 ns for an ion of mass 379.3). To maintain mass accuracy, the use of internal standards or external calibration performed on a membrane support was necessary. The immobilization of proteins on nylon membranes can be used to facilitate removal of water-soluble contaminants because the sample is retained when the membrane is immersed in water prior to adding the matrix solution. The feasibility of performing both chemical and enzymatic modifications of proteins adsorbed onto inert nylon supports prior to analysis by MALDi-MS is also demonstrated.     

Preliminary investigation of electrodynamic charged droplet processing to couple capillary liquid chromatography with matrix-assisted laser desorption/ionization mass spectrometry

Charged droplet processing methodology, that utilizes electrodynamic levitation technology to control the trajectories of picoliter volume charged droplets and deliver them to a target plate at atmospheric pressure, has been developed. Termed wall-less sample preparation (WaSP), this methodology offers several features that could prove beneficial to the preparation of sample spots from separation column effluents for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. These features include solute pre-concentration factors of 10(1) to 10(3) due to volatile solvent evaporation prior to droplet deposition onto the target plate, high spatial accuracy of the deposition position of each processed droplet (+/-5 microm), and the ability to prepare sample spots as small as 20 microm in diameter from a single droplet. Here a new mode of operation of this methodology is described and used as an offline post-column pre-concentrating interface between capillary liquid chromatography (capLC) and a target plate for offline MALDI-MS. Using a fraction from the capLC separation of peptides produced by the proteolytic digestion of the protein cytidine 5'-triphosphate:phosphocholine cytidylyltransferase, MALDI sample spots were prepared using the dried-droplet method, direct piezoelectric droplet dispensing, and the processing of piezo-dispensed droplets by WaSP. The sample spot morphology was investigated using light microscopy, and peptide ion abundances produced by MALDI were measured using time-of-flight (TOF) MS. The advantages of developing an online capLC/WaSP interface with MALDI-MS in the future are discussed along with some of the challenges that may be encountered in such an endeavor.     

Matching the laser wavelength to the absorption properties of matrices increases the ion yield in UV-MALDI mass spectrometry

A high analytical sensitivity in ultraviolet matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is only achieved if the laser wavelength corresponds to a high optical absorption of the matrix. Laser fluence and the physicochemical properties of the compounds, e.g., the proton affinity, also influence analytical sensitivity significantly. In combination, these parameters determine the amount of material ejected per laser pulse and the ion yield, i.e., the fraction of ionized biomolecules. Here, we recorded peptide ion signal intensities as a function of these parameters. Three cinnamic acid matrices were investigated: α-cyano-4-hydroxycinnamic acid, α-cyano-4-chlorocinnamic acid, and α-cyano-2,4-difluorocinnamic acid. In addition, 2,5-dihydroxybenzoic acid was used in comparison experiments. Ion signal intensities “per laser shot” and integrated ion signal intensities were acquired over 900 consecutive laser pulses applied on distinct positions on the dried-droplet sample preparations. With respect to laser wavelength, the two standard MALDI wavelengths of 337/355 nm were investigated. Also, 305 or 320 nm was selected to account for the blue-shifted absorption profiles of the halogenated derivatives. Maximal peptide ion intensities were obtained if the laser wavelength fell within the peak of the absorption profile of the compound and for fluences two to three times the corresponding ion detection threshold. The results indicate ways for improving the analytical sensitivity in MALDI-MS, and in particular for MALDI-MS imaging applications where a limited amount of material is available per irradiated pixel.     

Novel approach to enhance sensitivity in surface‐assisted laser desorption/ionization mass spectrometry imaging using deposited organic‐inorganic hybrid matrices

Sample pretreatment is key to obtaining good data in matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI). Although sublimation is one of the best methods for obtaining homogenously fine organic matrix crystals, its sensitivity can be low due to the lack of a solvent extraction effect. We investigated the effect of incorporating a thin film of metal formed by zirconium (Zr) sputtering into the sublimation process for MALDI matrix deposition for improving the detection sensitivity in mouse liver tissue sections treated with olanzapine. The matrix‐enhanced surface‐assisted laser desorption/ionization (ME‐SALDI) method, where a matrix was formed by sputtering Zr to form a thin nanoparticle layer before depositing MALDI organic matrix comprising α‐cyano‐4‐hydroxycinnamic acid (CHCA) by sublimation, resulted in a significant improvement in sensitivity, with the ion intensity of olanzapine being about 1800 times that observed using the MALDI method, comprising CHCA sublimation alone. When Zr sputtering was performed after CHCA deposition, however, no such enhancement in sensitivity was observed. The enhanced sensitivity due to Zr sputtering was also observed when the CHCA solution was applied by spraying, being about twice as high as that observed by CHCA spraying alone. In addition, the detection sensitivity of these various pretreatment methods was similar for endogenous glutathione. Given that sample preparation using the ME‐SALDI‐MSI method, which combines Zr sputtering with the sublimation method for depositing an organic matrix, does not involve a solvent, delocalization problems such as migration of analytes observed after matrix spraying and washing with aqueous solutions as sample pretreatment are not expected. Therefore, ME‐Zr‐SALDI‐MSI is a novel sample pretreatment method that can improve the sensitivity of analytes while maintaining high spatial resolution in MALDI‐MSI.     

Semiconductor applications of nanoliter droplet methodology with total reflection x-ray fluorescence analysis

In this study, the nanoliter dried spot method was applied to semiconductor contamination analysis to enhance vapor phase decomposition processes with total reflection X-ray fluorescence detection. Nanoliter-sized droplets (10 and 50 nl) were deposited onto native silicon oxide wafer surfaces in a clean room environment from both single and multielemental standards containing various concentrations of iron in different matrices. Direct comparisons were made to droplets formed by conventional VPD with similar iron standards. Nanoliter dried spots could be reproducibly deposited and dried in air with typical drying times ranging from 20 s to 2 min depending on the nanoliter volume deposited, compared to VPD spots which have drying times ranging from tens of minutes to several hours. Both types of residues showed a linear relationship between Fe intensity and mass deposited. Variable angle experiments showed that both nanoliter and VPD deposits of single element standards were film-like in character, while residues formed from much more complex matrices and higher mass loadings were particulate in character. For the experimental conditions used in this study (30 kV, 100 mA), typical TXRF spectral Fe limits of detection were calculated to be on the order of picograms or ∼1×10¹⁰ atoms/cm² for a 0.8 cm² X-ray excitation beam area for both nanoliter dried spots and VPD spots prepared from single elemental standards. Calculated Fe detection limits for 200 mm diameter silicon wafers used in this study were in the ∼1×10⁸ atoms/cm² range. By using nanoliter sized droplets, the required sample volume is greatly reduced resulting in higher sample throughput than with conventional VPD methods.     

Sensitive detection of phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry: use of alkylphosphonic acids as matrix additives

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been one of the most powerful tools for analyzing protein phosphorylation. However, it is frequently difficult to detect phosphopeptides with high sensitivity by MALDI-MS. In our investigation of matrix/matrix-additive substances for improving the phosphopeptide ion response in MALDI-MS, we found that the addition of low-concentration alkylphosphonic acid to the matrix/analyte solution significantly enhanced the signal of phosphopeptides. In this study, the combination of methanediphosphonic acid and 2,5-dihydroxybenzoic acid gave the best results. In addition to enhancing the signal of the phosphopeptides, alkylphosphonic acid almost completely eliminated the signals of sodium and potassium ion adducts. We report herein sensitive detection of phosphopeptides by MALDI-MS with the use of alkylphosphonic acids as matrix additives.