Capillary electrophoresis--matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a vacuum deposition interface

An improved vacuum deposition interface for coupling capillary electrophoresis with MALDI-TOF MS has been developed. Liquid samples consisting of analyte and matrix were deposited on a moving tape in the evacuated source chamber of a TOF mass spectrometer, enabling 24 h of uninterrupted analysis. The vacuum deposition procedure was compared with the dried-droplet method, and it was found that vacuum deposition generated significantly more reproducible signal intensity, eliminating the need for "sweet spot" searching. A concentration detection limit in the low-nanomolar range has been achieved with a low-attomole amount of sample consumed per spectrum. In addition, ion suppression caused by hydrophobicity differences in the analytes was reduced. To minimize ion suppression further, separation prior to MALDI MS analysis was employed. The performance of capillary electrophoresis (CE)-MALDI-TOF MS using the vacuum deposition interface was evaluated with a peptide mixture injected at low-femtomole levels. All peptides were baseline resolved with separation efficiencies in the range of 250000-400000 plates/m (2-3-s band half-width), demonstrating the high separation efficiency of the CE-MALDI MS coupling. A fast (approximately 40 s) CE separation of a mixture of angiotensins was found to reduce significantly ion suppression and enable trace level detection. It was also shown, for the analysis of an enolase digest, that sequence coverage of 65% was obtained using CE separation compared to 52% using step-elution solid-phase extraction and 44% in the control experiment using an unseparated mixture.     

Enhanced neuropeptide profiling via capillary electrophoresis off-line coupled with MALDI FTMS

An off-line interface incorporating sheathless flow and counter-flow balance is developed to couple capillary electrophoresis (CE) to matrix-assisted laser desorption ionization Fourier transform mass spectrometry (MALDI FTMS) for neuropeptide analysis of complex tissue samples. The new interface provides excellent performance due to the integration of three aspects: (1) A porous polymer joint constructed near the capillary outlet for the electrical circuit completion has simplified the CE interface by eliminating a coaxial sheath liquid and enables independent optimization of separation and deposition. (2) The electroosmotic flow at reversed polarity (negative) mode CE is balanced and reversed by a pressure-initiated capillary siphoning (PICS) phenomenon, which offers improved CE resolution and simultaneously generates a low flow (<100 nL/min) for fraction collection. (3) The predeposited nanoliter volume 2,5-dihydroxybenzoic acid (DHB) spots on a Parafilm-coated MALDI sample plate offers an improved substrate for effective effluent enrichment. Compared with direct MALDI MS analysis, CE separation followed by MALDI MS detection consumes nearly 10-fold less sample (50 nL) while exhibiting 5-10-fold enhancement in S/N ratio that yields the limit of detection down to 1.5 nM, or 75 attomoles. This improvement in sensitivity allows 230 peaks detected in crude extracts from only a few pooled neuronal tissues and increases the number of identified peptides from 19 to 43 (Cancer borealis pericardial organs (n = 4)) in a single analysis. In addition, via the characteristic migration behaviors in CE, some specific structural and chemical information of the neuropeptides such as post-translational modifications and family variations has been visualized, making the off-line CE-MALDI MS a promising strategy for enhanced neuropeptidomic profiling.     

Advancing matrix-assisted laser desorption/ionization-mass spectrometric imaging for capillary electrophoresis analysis of peptides

In this work, the utilization of matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) for capillary electrophoresis (CE) analysis of peptides based on a simple and robust off-line interface has been investigated. The interface involves sliding the CE capillary distal end within a machined groove on a MALDI sample plate, which is precoated with a thin layer of matrix for continuous sample deposition. MALDI-MSI by time of flight (TOF)/TOF along the CE track enables high-resolution and high-sensitivity detection of peptides, allowing the reconstruction of a CE electropherogram while providing accurate mass measurements and structural identification of molecules. Neuropeptide standards and their H/D isotopic formaldehyde-labeled derivatives were analyzed using this new platform. Normalized intensity ratios of individual ions extracted from the CE trace were compared to MALDI-MS direct analysis and the theoretical ratios. The CE-MALDI-MSI results show potential for sensitive and quantitative analysis of peptide mixtures spanning a wide dynamic range.     

Two-layer sample preparation method for MALDI mass spectrometric analysis of protein and peptide samples containing sodium dodecyl sulfate

Sodium dodecyl sulfate (SDS) is widely used in protein sample workup. However, many mass spectrometric methods cannot tolerate the presence of this strong surfactant in a protein sample. We present a practical and robust technique based on a two-layer matrix/sample deposition method for the analysis of protein and peptide samples containing SDS by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The two-layer method involves the deposition of a mixture of sample and matrix on top of a thin layer of matrix crystals. It was found that for SDS-containing samples, the intensity of the MALDI signals can be affected by the conditions of sample preparation: on-probe washing, choice of matrix, deposition method, solvent system, and protein-to-SDS ratio. However, we found that, under appropriate conditions, the two-layer method gave reliable MALDI signals for samples with levels of SDS up to approximately 1%. The applications of this method are demonstrated for MALDI analysis of hydrophobic membrane proteins as well as bacterial extracts. We envision that this two-layer method capable of handling impure samples including those containing SDS will play an important role in protein molecular weight analysis as well as in proteome identification by MALDI-MS and MS/MS.     

Online CE-MALDI-TOF MS using a rotating ball interface

We report on the construction and performance of a rotating ball interface for online coupling of capillary electrophoresis (CE) to matrix-assisted laser desorption ionization (MALDI) mass spectrometry with a time-of-flight (TOF) mass analyzer. The interface is based on a rotating stainless steel ball that transports samples from atmospheric pressure to the high vacuum of the mass spectrometer for desorption and ionization. The sample is deposited directly from a 50-microm-i.d. separation capillary onto the 19-mm ball that is rotating at 0.03 to 0.3 rpm. The sample is mixed online with matrix flowing from a separate 50-microm-i.d. capillary. The sample deposit dries before it is rotated past a polymer gasket and into the laser ionization region. Cleaning of the interface is accomplished using solvent-saturated felt, which cleans the ball surface after it rotates out of the ionization chamber. On-line CE-MALDI is demonstrated, and the performance is evaluated with the analysis of a mixture of three peptides: [Lsy8] vasopressin, substance P, and neurotensin. The rotating ball interface to MALDI-TOF MS demonstrated mass detection limit in the high femtomole range. The interface has negligible memory effect and shows no significant electrophoretic peak broadening when operated under optimized conditions.     

2,5-Dihydroxybenzoic acid butylamine and other ionic liquid matrixes for enhanced MALDI-MS analysis of biomolecules

The performance of the new ionic liquid MALDI-MS matrix 2,5-dihydroxybenzoic acid butylamine (DHBB) was assessed and compared to results obtained with the ionic liquid MALDI-MS matrixes alpha-cyano-4-hydroxycinnamic acid butylamine (CHCAB), 3,5-dimethoxycinnamic acid triethylamine (SinTri), and the frequently used solid MALDI matrixes 2,5-dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA). The vacuum-stable, liquid consistency of ionic liquid matrix sample preparations considerably enhanced MALDI-MS analysis in terms of shot-to-shot reproducibility. Consequently, relative standard deviations serving as a measure for reproducibility of intensity-values acquired from 90 different spots on one MALDI-MS preparation were approximately one-half as high when solid DHB was replaced by the ionic liquid DHBB and eight times lower after exchange of solid CHCA by ionic liquid CHCAB. Interestingly, the ionic liquid MALDI matrix DHBB conserved the broad applicability of its solid analogue DHB, reduced MALDI induced fragmentation of monosialylated glycans and gangliosides, and was the superior ionic liquid matrix for MALDI-MS analysis of oligosaccharides and polymers, such as poly(ethylene glycol). It also worked well with glycoconjugates, peptides, and proteins; however, the tendency of DHBB to form multiple alkali adduct ions with peptides and proteins made CHCAB the ionic liquid matrix of choice for peptides. SinTri was the best ionic liquid matrix for proteins of high molecular weight, such as IgG. Furthermore, it was demonstrated for the first time that solvent properties and MALDI matrix properties of ionic liquids, such as DHBB, can be combined to enable fast, direct screening of an enzymatic reaction. This was proven by the desialylation of sialylactose with sialidase from Clostridium perfringens in the presence of diluted aqueous DHBB and subsequent direct MALDI-MS analysis of the reaction mixture.     

Coupling of protein HPLC to MALDI-TOF MS using an on-target device for fraction collection, concentration, digestion, desalting, and matrix/analyte cocrystallization

Multidimensional protein chromatography offers an alternative to gel-based separations for large-scale proteomic analyses of highly complex mixtures. However, these liquid separations divide the original mixtures into multitudes of discrete samples, each of which may require numerous steps of sample manipulation, such as fraction collection, buffer exchange, protease digestion, peptide desalting, and, in the case of MALDI-MS, matrix and analyte cocrystallization on target. When traditional high-flow liquid chromatography is used, large volumes of solvent must also be removed from fractions to maximize MS sensitivity. Although robotic liquid-handling devices can facilitate these steps and reduce analyst/sample contact, they remain prototypic and expensive. Here, we explore the use of a novel, one-piece elastomeric device, the BD MALDI sample concentrator, which affixes to a MALDI target to create a prestructured 96-well sample array on the target surface. We have developed methodologies to process high-flow HPLC fractions by collecting them directly into the elastomeric device and then subjecting them to sequential on-target sample concentration, buffer exchange, digestion, desalting, and matrix/analyte cocrystallization for MALDI-MS analyses. We demonstrate that this methodology enables the rapid digestion and analysis of low amounts of proteins and that it is effective in the characterization of an HPLC-fractionated protein mixture by MALDI-TOF MS followed by peptide mass fingerprinting.     

Surfactant-aided, matrix-assisted laser desorption/ionization mass spectrometry of hydrophobic and hydrophilic peptides

The analysis of hydrophobic and hydrophilic peptides in an aqueous medium using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported. The key development allowing for simultaneous analysis of both hydrophobic and hydrophilic components of the sample mixture is the use of surfactants to solubilize the hydrophobic components in the MALDI matrix solution. A wide variety of anionic, cationic, zwitterionic, and nonionic surfactants were evaluated for their ability to assist in the generation of an abundant pseudomolecular ion from a model hydrophobic peptide ([tert-butoxycarbonyl]Glu[gamma-O-benzyl]-Ala-Leu-Ala[O-phenacyl ester]). The results indicate that the most successful surfactant among those studied for analyzing the model hydrophobic peptide is sodium dodecyl sulfate (SDS). SDS exhibited no interfering surfactant background ions, little to no loss of the acid-labile protecting groups from the model hydrophobic peptide, and an abundant pseudomolecular ion of the analyte. In addition, the use of surfactants is shown to be compatible with hydrophilic peptides as well. Mixtures of hydrophobic and hydrophilic peptides were characterized using surfactant-aided (SA) MALDI-MS, and it is demonstrated that all components are detectable once the surfactant is included in the sample solution. We conclude that the key benefit of using SA-MALDI-MS is its ability to simultaneously analyze hydrophobic and hydrophilic peptides from a single sample mixture, including synthetic peptides containing acid- and base-labile protecting groups.     

Impulse-driven heated-droplet deposition interface for capillary and microbore LC-MALDI MS and MS/MS

An automated off-line liquid chromatography-matrix-assisted laser desorption ionization (LC-MALDI) interface capable of coupling both capillary and microbore LC separations with MALDI mass spectrometry (MS) and tandem mass spectrometry (MS/MS) has been developed. The interface is a combination of two concepts: analyte concentration from heated hanging droplets and impulse-driven droplet deposition of LC fractions onto a MALDI sample plate. At room temperature the interface allows the coupling of capillary LC separations (i.e., flow rate of <5 microL/min) with MALDI MS. With heating, it can be used to combine microbore LC operated at a relatively high flow rate of up to 50 microL/min with MALDI MS. The collected fractions can be analyzed by MALDI MS and MS/MS instruments, such as time-of-flight (TOF) and quadrupole-TOF MS. Performance of the interface was examined using several peptide and protein standards. It was shown that, using MALDI-TOF MS, [GLU1]-fibrinopeptide B could be detected with a total injection amount of 5 fmol to microbore LC. Chromatographic performance was also monitored. A peak width of 12 s at half-height for [GLU1]-fibrinopeptide B showed no evidence of band broadening due to the interface. The ability of the interface to mitigate ion suppression was studied using a mixture of 100 fmol of [GLU1]-fibrinopeptide B and 10 pmol of cytochrome c tryptic digest. Although fully suppressed under direct MALDI conditions, LC-MALDI analysis was able to detect the 100 fmol peptide with 10 s fraction collection. Finally, the ability to inject relatively large sample amounts to improve detectability of low-abundance peptides was illustrated in the analysis of phosphopeptides from alpha-casein tryptic digests. A digest loaded on column to 2.4 microg and analyzed by LC-MALDI MS/MS resulted in 82% sequence coverage and detection of all nine phosphoserine residues. It is concluded that, being able to handle both high- and low-flow LC separations, the impulse-driven heated-droplet interface provides the flexibility to carry out MALDI analysis of peptides and proteins depending on the information sought after, analysis speed, and sample size.     

Functional microfabricated sample targets for matrix-assisted laser desorption/ionization mass spectrometry analysis of ribonucleic acids

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful analytical tool for the structural characterization of proteins and nucleic acids. However, many proteomics or genomics methodologies that employ MALDI-MS require external sample manipulation, which limits the overall throughput of analysis. We have focused on fabricating functional MALDI sample plates that would permit the on-probe characterization of nucleic acids. Here, we present results arising from the fabrication of functional sample plates composed of poly(methyl methacrylate) (PMMA). The PMMA sample plates were fabricated by a CNC milling technique. The key structural feature of our microfabricated samples plates is the presence of individual cylindrical posts (360 microm x 360 microm), which serve as individual sample targets within the overall PMMA-based MALDI sample plate. Functionality is added to these microposts via the covalent attachment of enzymes. As an example of the applicability of these microfabricated sample plates, enzymatic digestion of ribonucleic acids was performed on probe (i.e., on the micropost) with subsequent analysis by MALDI-MS. Advantages to such an approach include a reduction in sample handling (and concomitant sample losses) and a reduction in the amount of sample required for analysis due to the small surface area of the microposts.     

Enhancement of MALDI-MS spectra of C-terminal peptides by the modification of proteins via an active ester generated in situ from an oxazolone

For selective C-terminal derivatization of peptides and proteins, we have devised a method for activating the C-terminal carboxyl group by extending the oxazolone chemistry. A mixture of formic acid and acetic anhydride was found to be effective for the formation of an oxazolone, which was converted to an active ester in situ in the presence of a phenol or an N-hydroxide. In particular, the resulting active ester with pentafluorophenol facilitated the subsequent reaction with an amine and the hydrazine derivative to yield the C-terminal amide and hydrazide, respectively. The peptides thus coupled with arginine methyl ester or 2-hydrazino-2-imidazoline containing the guanidino moiety exhibited the positive-ion peaks in matrix-assisted laser desorption/ionization (MALDI) mass spectra with appreciably enhanced intensities. As expected from the reaction mechanism, the carboxyl groups of aspartic and glutamic acid residues were not modified, while the amino groups that could react with the activated peptides were concomitantly protected by formylation. The MALDI peaks corresponding to the C-terminal peptide fragments of proteins were specifically enhanced, discriminating against those from internal peptides that were not tagged with a positive charge. In favorable cases, the C-terminal peptide fragments were clearly discerned by MALDI-MS after chymotryptic digestion and were identified by their MALDI postsource decay analysis. Based on these results, we suggest a method for C-terminal sequencing of a protein.     

Nanoliter-volume selective sampling of peptides based on isoelectric points for MALDI-MS

A simple way to selectively isolate peptides based on their isoelectric points (pI) for MALDI mass spectral analysis is described. An applied voltage was used to electromigrate peptides into a capillary. The capillary was modified with a zwitterionic surfactant, 1,2-dilauroyl-sn-phosphatidylcholine (DLPC), to suppress the electroosmotic flow (EOF) during injection. Hence, either the cationic or the anionic peptides were introduced, depending on the voltage polarity. By controlling the pH, selective loading of peptides was performed to isolate trace components from a mixture. The injected sample plugs were subsequently spotted in nanoliter volumes for MALDI-MS analysis. No significant sample losses resulting from selective sampling were detected. Low attomole-level detection of peptides (adrenocorticotropic hormone fragment 18-39, pI 4.25) was achieved from a mixture containing other peptides (angiotensin I, pI 6.92, and bradykinin, pI 12.00) at 100 000-fold higher concentrations.     

Combining liquid chromatography with MALDI mass spectrometry using a heated droplet interface

A novel interfacing technology is described to combine solution-based separation techniques such as liquid chromatography (LC) with matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The interface includes a transfer tube having an inlet and an outlet, the inlet being adapted to accept the LC effluents and the outlet being adapted to form continuously replaced, hanging droplets of the liquid stream, and a MALDI sample plate mounted below the outlet of the transfer tube for collecting the droplets. The liquid stream in the transfer tube is heated to a temperature sufficient to cause partial evaporation of the carrier solvent from the hanging droplets. The droplets are dislodged to the MALDI plate, which is heated to above the boiling point of the carrier solvent to cause further evaporation of the carrier solvent from the collected droplets. It is found that analytes can be fractionated and deposited to a sample spot of 0.8 mm in diameter when a liquid flow rate of up to 50 microL/min and a fractionation interval of 1 min/spot are used. Flow rate of up to 200 microL/min can be used with a deposition sample spot of 2.4 mm in diameter on a commercial MALDI target. This heated droplet interface does not introduce sample loss, and the detection sensitivity of LC/MALDI is similar to that of standard MALDI, i.e., low femtomoles for peptide analysis with a microliter sample deposition. It is compatible with microbore and narrow-bore column separation, thus allowing the injection of a larger amount of sample for separation and analysis, compared to a capillary column LC/MALDI system. The detection dynamic range is shown to be in the order of 10(6) for peptide mixture analysis, which is 4 orders of magnitude greater than standard MALDI. The application of this interface for combining LC with MALDI MS/MS is demonstrated in the proteome analysis of water-soluable protein components of E. coli K12 extracts.     

Peptide profiling of cells with multiple gene products: combining immunochemistry and MALDI mass spectrometry with on-plate microextraction

Due to the intracellular chemical complexity and a wide range of transmitter concentrations, the detection of the complete set of peptide transmitters in a single cell is problematic. In the current study, a multidisciplinary approach combining single-cell MALDI-MS peptide profiling, northern analysis, in situ hybridization, and immunocytochemistry allows characterization of a more complete set of neurotransmitters than individual approaches in the Aplysia californica B1 and B2 motor neurons. Because different results were obtained using both in situ and immunohistochemical techniques compared to previous reports, MALDI-MS assays have been used to examine CP1-related gene products in these cells. However, MALDI with standard sample preparation does not detect the presence of the CP1 gene products. A novel on-plate microextraction approach using concentrated MALDI matrix 2,5-dihydroxybenzoic acid with a mixture of acetone and water as the solvent has been developed to allow the detection of trace-level gene expression products. Both neuropeptide precursors in the B1 and B2 neurons-the SCP and CP1 prohormones-end with large peptides that have multiple cysteine residues. For SCP, MALDI-MS verifies the presence of a novel 9325 Da SCP-related peptide. In the case of CP1, a disulfide-bonded homodimer is detected and the disulfide bonding pattern elucidated using MALDI-MS coupled with on-plate enzymatic digestion.     

Pressure-assisted capillary electrophoresis coupling with matrix-assisted laser desorption/ionization-mass spectrometric imaging for quantitative analysis of complex Peptide mixtures

Herein, we report a pressure-assisted capillary electrophoresis-mass spectrometric imaging (PACE-MSI) platform for peptide analysis. This new platform has addressed the sample diffusion and peak splitting problems that appeared in our previous groove design, and it enables homogeneous deposition of the CE trace for high-throughput MALDI imaging. In the coupling of CE to MSI, individual peaks (m/z) can be visualized as discrete colored image regions and extracted from the MS imaging data, thus eliminating issues with peak overlapping and reducing reliance on an ultrahigh mass resolution mass spectrometer. Through a PACE separation, 46 tryptic peptides from bovine serum albumin and 150 putative neuropeptides from the pericardial organs of a model organism blue crab Callinectes sapidus were detected from the MALDI MS imaging traces, enabling a 4- to 6-fold increase of peptide coverage as compared with direct MALDI MS analysis. For the first time, quantitation with high accuracy was obtained using PACE-MSI for both digested tryptic peptides and endogenous neuropeptides from complex biological samples in combination with isotopic formaldehyde labeling. Although MSI is typically employed in tissue imaging, we show in this report that it offers a unique tool for quantitative analysis of complex trace-level analytes with CE separation. These results demonstrate a great potential of the PACE-MSI platform for enhanced quantitative proteomics and neuropeptidomics.     

Suppression of alpha-cyano-4-hydroxycinnamic acid matrix clusters and reduction of chemical noise in MALDI-TOF mass spectrometry

Progress in high-throughput MALDI-TOFMS analysis, especially in proteome applications, requires development of practical and efficient procedures for the preparation of proteins and peptides in a form suitable for high acquisition rates. These methods should improve successful identification of peptides, which depends on the signal intensity and the absence of interfering signals. Contamination of MALDI samples with alkali salts results in reduced MALDI peptide sensitivity and causes matrix cluster formation (widely reported for CHCA matrix) observed as signals dominating in the range below m/z 1200 in MALDI spectra. One way to remove these background signals, especially for concentrations of peptides lower than 10 fmol/microL, is to wash matrix/sample spots after peptide cocrystallization on the MALDI plate with deionized water prior to analysis. This method takes advantage of the low water solubility of the CHCA compared to its alkali salts. We report here that the application of some ammonium salt solutions, such as citrates and phosphates, instead of deionized water greatly improves the efficiency of this washing approach. Another way to reduce matrix cluster formation is to add ammonium salts as a part of the MALDI matrix. The best results were obtained with monoammonium phosphate, which successfully suppressed matrix clusters and improved sensitivity. Combining both of these approaches-the addition of ammonium salts in the CHCA matrix followed by one postcrystallization washing step with ammonium buffer-provided a substantial ( approximately 3-5-fold) improvement in the sensitivity of MALDI-MS detection compared to unwashed sample spots. This sample preparation method resulted in improved spectral quality and was essential for successful database searching for subnanomolar concentrations of protein digests.     

Direct analysis of an oligomeric hindered amine light stabilizer in polypropylene materials by MALDI-MS using a solid sampling technique to study its photostabilizing action

A novel method for the direct analysis of small amounts of an oligomeric hindered amine light stabilizer (HALS) occluded in polypropylene (PP) material was developed to study its photostabilizing action on the basis of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using a solid sampling technique while avoiding troublesome solvent extraction. In this sampling protocol, the powdered mixture of PP composite sample containing trace amounts of an oligomeric HALS, Adekastab LA-68LD (MW = 1900), and the matrix reagent (dithranol) was spotted on the sample plate, then ion exchanged water was deposited onto the mixture to make a suspension, and finally, the dried mixture adhered on the plate was subjected to MALDI-MS measurement. On the mass spectrum thus obtained by the solid sampling MALDI, the molecular ions of the HALS desorbed from the PP composite were clearly observed as three major series of the HALS components in the range up to about m/z 7000 with little interference by the PP substrate and the other additives. Moreover, in the MALDI-MS spectra for the UV-exposed sample, the satellite peaks around the major HALS components proved to enhance significantly, reflecting the oxidized HALS species at the tetramethylpiperidine units to cause the photostabilizing action. In addition, hydrolyzed HALS species were also observed for the irradiated sample. These results suggest that not only the oxidation reaction but also the hydrolysis or decomposition of the oligomeric HALS components competitively proceed in the PP composites during UV exposure.     

Identification of individual proteins in complex protein mixtures by high-resolution, high-mass-accuracy MALDI TOF-mass spectrometry analysis of in-solution thermal denaturation/enzymatic digestion

Identification of individual proteins in complex protein mixtures by high-resolution (HR), high-mass-accuracy matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) is demonstrated for synthetic protein mixtures. Instead of chemical denaturation, thermal denaturation followed by in-solution trypsin digestion is used to achieve uniform digestion of the constituents of the protein mixture. Protein identification is carried out using protein database searches with search scoring systems, which seems more effective than conventional peptide mass mapping without using a scoring system. Identification of individual proteins by MALDI HR-TOF-MS peptide mass mapping dramatically reduces data acquisition/analysis time and does not require special equipment for sample preparation/transfer prior to mass spectral analysis.     

An automated noncontact deposition interface for liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry

A new multichannel deposition system was developed for off-line liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry (LC/MALDI-MS). This system employs a pulsed electric field to transfer the eluents from multiple parallel columns directly onto MALDI targets without the column outlets touching the target surface. The deposition device performs well with a wide variety of solvents that have different viscosities, vapor pressures, polarities, and ionic strengths. Surface-modified targets were used to facilitate concentration and precise positioning of samples, allowing for efficient automation of high-throughput MALDI analysis. The operational properties of this system allow the user to prepare samples using MALDI matrixes whose properties range from hydrophilic to hydrophobic. The latter, exemplified by alpha-cyano-4-hydroxycinnamic acid, were typically processed with a multistep deposition method consisting of precoating of individual spots on the target plate, sample deposition, and sample recrystallization steps. Using this method, 50 amol of angiotensin II was detected reproducibly with high signal-to-noise ratio after LC separation. Experimental results show that there is no significant decrease in chromatographic resolution using this device. To assess the behavior of the apparatus for complex mixtures, 5 microg of a tryptic digest of the cytosolic proteins of yeast was analyzed by LC/MALDI-MS and more than 13,500 unique analytes were detected in a single LC/MS analysis.     

Patterned monolayer/polymer films for analysis of dilute or salt-contaminated protein samples by MALDI-MS

This paper describes a surface science/mass spectrometry effort to develop and characterize a patterned gold surface that serves as a MALDI sample platform capable of concentrating and purifying proteins. Using microcontact printing, small (200-microm diameter) hydrophilic spots of bare gold or chemically anchored poly(acrylic acid) (PAA) are patterned at 5-mm intervals in a hydrophobic field consisting of a self-assembled monolayer of hexadecanethiol. Building on recent innovations by others, the small hydrophilic spots concentrate the sample to achieve good reproducibility and high sensitivity in the MALDI signal. One of the key features in this work is the combination of the high density of carboxylate groups in PAA with a small spot size to afford both concentration and purification of proteins via ionic interactions. This translates into detection limits for salt-contaminated proteins that are 20-100 times lower (low femtomole) than those reported for previous polymer- or monolayer-modified MALDI probes (using proteins in the 3-15-kDa range). Reflectance FT-IR spectroscopy and ellipsometry were used to determine the amount of protein adsorbed to a PAA-modified sample plate as a function of pH and salt concentration. Amide absorbances in IR spectra correlate well with MALDI-MS signals measured after addition of 2,5-dihydroxybenzoic acid as a matrix.