Development and characterization of an ionization technique for analysis of biological macromolecules: liquid matrix-assisted laser desorption electrospray ionization

We have developed an atmospheric pressure ionization technique called liquid matrix-assisted laser desorption electrospray ionization (liq-MALDESI) for the generation of multiply charged ions by laser desorption from liquid samples deposited onto a stainless steel sample target biased at a high potential. This variant of our previously reported MALDESI source does not utilize an ESI emitter to postionize neutrals. Conversely, we report desorption and ionization from a macroscopic charged droplet. We demonstrate high mass resolving power single-acquisition FT-ICR-MS analysis of peptides and proteins ranging from 1 to 8.6 kDa at atmospheric pressure. The liquid sample acts as a macroscopic charged droplet similar to those generated by electrospray ionization, whereby laser irradiation desorbs analyte from organic matrix containing charged droplets generating multiply charged ions. We have observed a singly charged radical cation of an electrochemically active species indicating oxidation occurs for analytes and therefore water; the latter would play a key role in the mechanism of ionization. Moreover, we demonstrate an increase in ion abundance and a concurrent decrease in surface tension with an increase in the applied potential.     

Sample depletion of the matrix-assisted laser desorption process monitored using radionuclide detection

To investigate analyte consumption during the laser desorption process, matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is combined with radionuclide detection. Radionuclide detection provides highly sensitive and quantitative information on the amount of radiolabeled analytes in a MALDI MS sample spot. 14C-Labeled cytochrome c is deposited with 2,5-dihydroxybenzoic acid in 10-nL volume spots. By comparing radioactivity levels of the labeled cytochrome c both before and after spectral acquisition, the reduction in labeled analyte molecules on the target allows monitoring of the moles of desorbed sample. Through a depletion study on this sample, the amount of analyte consumed for MALDI time-of-flight spectral acquisition and the average number of molecules desorbed per laser ablation are determined. When [14C]-cytochrome c is no longer detected by MALDI MS, approximately 70% of the original analyte remains in the sample spots. Redissolving the spots produced further desorption, indicating that the analyte before dissolution was in a physical environment that did not facilitate the desorption process. As a technique with a response that does not depend on the environment of the analyte, radionuclide detection allows characterization of mass-limited sampling methods to better understand the MALDI process.     

In situ liquid-liquid extraction as a sample preparation method for matrix-assisted laser desorption/ionization MS analysis of polypeptide mixtures

A novel liquid-liquid extraction (LLE) procedure was investigated for preparation of peptide and protein samples for matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). LLE using ethyl acetate as the water-immiscible organic solvent enabled segregation of hydrophobic and hydrophilic polypeptides in mixtures, thereby reducing the complexity of mass spectra obtained by MALDI MS. The LLE technique was optimized for rapid and sensitive in situ (on-target) sample preparation for MALDI MS analysis of proteins and peptides at low-picomole and subpicomole levels. Addition of MALDI matrix to the organic solvent enhanced the efficiency of the LLE-MALDI MS method for analysis of hydrophobic peptides and proteins. LLE-MALDI MS enabled the detection of the hydrophobic membrane protein bacteriorhodopsin as a component in a simple protein mixture. Peptide mixtures containing phosphorylated, glycosylated, or acylated peptides were successfully separated and analyzed by the in situ LLE-MALDI MS technique and demonstrate the potential of this method for enhanced separation and structural analysis of posttranslationally modified peptides in proteomics research.     

High sensitivity and analyte capture with desorption/ionization mass spectrometry on silylated porous silicon

Silylation chemistry on porous silicon provides for ultrahigh sensitivity and analyte specificity with desorption/ionization on silicon mass spectrometry (DIOS-MS) analysis. Here, we report that the silylation of oxidized porous silicon offers a DIOS platform that is resistant to air oxidation and acid/base hydrolysis. Furthermore, surface modification with appropriate hydrophobic silanes allows analytes to absorb to the surface via hydrophobic interactions for direct analyte extraction from complex matrixes containing salts and other nonvolatile interferences present in the sample matrix. This enables rapid cleanup by simply spotting the sample onto the modified DIOS target and removing the liquid phase containing the interferences. This approach is demonstrated in the analysis of protein digests and metabolites in biofluids, as well as for the characterizing of inhibitors from their enzyme complex. An unprecedented detection limit of 480 molecules (800 ymol) for des-Arg(9)-bradykinin is reported on a pentafluorophenyl-functionalized DIOS chip.     

Quantitative matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis of synthetic polymers and peptides

Matrix-assisted laser desorption ionization (MALDI) is a very powerful and widely used mass spectrometric technique to ionize high molecular weight compounds. The most commonly used dried droplet (DD) technique can lead to a concentration distribution of the analyte on the target and is therefore often not suitable for reproducible analyses. We developed a new solvent-free deposition technique, called compressed sample (CS), to prevent the distribution of the analytes caused by the crystallization of the compounds. The CS technique presented in this work allows the quantitative analysis of synthetic polymers such as derivatized maltosides with correlation coefficients of 0.999 and peptides up to 3500 Da with correlation coefficients of at least 0.982 without the use of stable-isotope-labeled standards.     

Targeted analyte detection by standard addition improves detection limits in matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) has proven an effective tool for fast and accurate determination of many molecules. However, the detector sensitivity and chemical noise compromise the detection of many invaluable low-abundance molecules from biological and clinical samples. To challenge this limitation, we developed a targeted analyte detection (TAD) technique. In TAD, the target analyte is selectively elevated by spiking a known amount of that analyte into the sample, thereby raising its concentration above the noise level, where we take advantage of the improved sensitivity to detect the presence of the endogenous analyte in the sample. We assessed TAD on three peptides in simple and complex background solutions with various exogenous analyte concentrations in two MALDI matrices. TAD successfully improved the limit of detection (LOD) of target analytes when the target peptides were added to the sample in a concentration close to optimum concentration. The optimum exogenous concentration was estimated through a quantitative method to be approximately equal to the original LOD for each target. Also, we showed that TAD could achieve LOD improvements on an average of 3-fold in a simple and 2-fold in a complex sample. TAD provides a straightforward assay to improve the LOD of generic target analytes without the need for costly hardware modifications.     

Functional Femtoliter Droplets for Ultrafast Nanoextraction and Supersensitive Online Microanalysis

A universal femtoliter surface droplet‐based platform for direct quantification of trace of hydrophobic compounds in aqueous solutions is presented. Formation and functionalization of femtoliter droplets, concentrating the analyte in the solution, are integrated into a simple fluidic chamber, taking advantage of the long‐term stability, large surface‐to‐volume ratio, and tunable chemical composition of these droplets. In situ quantification of the extracted analytes is achieved by surface‐enhanced Raman scattering (SERS) spectroscopy by nanoparticles on the functionalized droplets. Optimized extraction efficiency and SERS enhancement by tuning droplet composition enable quantitative determination of hydrophobic model compounds of rhodamine 6G, methylene blue, and malachite green with the detection limit of 10^?9 to 10^?11 m and a large linear range of SERS signal from 10^?9 to 10^?6 m of the analytes. The approach addresses the current challenges of reproducibility and the lifetime of the substrate in SERS measurements. This novel surface droplet platform combines liquid–liquid extraction and highly sensitive and reproducible SERS detection, providing a promising technique in current chemical analysis related to environment monitoring, biomedical diagnosis, and national security monitoring.     

Solvent-free MALDI-MS for the analysis of a membrane protein via the mini ball mill approach: case study of bacteriorhodopsin

A mini ball mill (MBM) solvent-free matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) method allows for the analysis of bacteriorhodopsin (BR), an integral membrane protein that previously presented special analytical problems. For well-defined signals in the molecular ion region of the analytes, a desalting procedure of the MBM sample directly on the MALDI target plate was used to reduce adduction by sodium and other cations that are normally attendant with hydrophobic peptides and proteins as a result of the sample preparation procedure. Mass analysis of the intact hydrophobic protein and the few hydrophobic and hydrophilic tryptic peptides available in the digest is demonstrated with this robust new approach. MS and MS/MS spectra of BR tryptic peptides and intact protein were generally superior to the traditional solvent-based method using the desalted "dry" MALDI preparation procedure. The solvent-free method expands the range of peptides that can be effectively analyzed by MALDI-MS to those that are hydrophobic and solubility-limited.     

Kinetics and the on-site application of standards in a solid-phase microextraction fiber

The kinetics of the desorption of analytes from a SPME fiber into an agitated sample matrix was studied, and a theoretical model was proposed to describe the dynamic desorption process, based on the steady-state diffusion of analytes in the extraction phase and in the boundary layer. It was found that the desorption of analytes from a SPME fiber into an agitated sampling matrix is isotropic to the absorption of the analytes onto the SPME fiber from the sample matrix under the same agitation conditions, and this allows for the calibration of absorption using desorption. The calibration was accomplished by exposing a SPME fiber, preloaded with a standard, to an agitated sample matrix, during which desorption of the standard and absorption of analytes occurred simultaneously. When the standard was the isotopically labeled analogue of the target analyte, the information from the desorption process, i.e., time constant a, could be directly used for estimating the concentration of the target analyte. When the standard varied from the target analyte, the mass-transfer coefficient of the analyte could be extrapolated from that of the standard. These predictions agree well with experimental results. This approach facilitates the full integration of sampling, sample preparation, and sample introduction, especially for on-site or in vivo investigations, where the addition of standards to the sample matrix, or control of the velocity of the sample matrix, is very difficult.     

Confocal Fluorescence Microscopic Imaging for Investigating the Analyte Distribution in MALDI Matrices

The analytical performance of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is strongly influenced by the method of analyte and matrix preparation. We report a nonintrusive method based on laser confocal microscopic imaging technology to examine the MALDI samples prepared by various protocols. In this method, the analyte is tagged with a fluorescent group. The matrix and analyte are prepared under the same conditions as those used in conventional MALDI experiments. It is demonstrated that confocal microscopy can provide clear, three-dimensional images of sample crystals as well as the analyte distribution within the crystals. It is shown that the analyte is incorporated into the matrix crystals for all the sample preparation protocols examined. Moreover, the confocal microscopic images reveal that, with the use of a dried-droplet method for sample/matrix preparation, the analyte is not uniformly distributed within the matrix crystals. In some crystals, no analyte is incorporated. In addition, it is found that large crystals formed using a slow growth process display a more uniform analyte distribution. Relatively more uniform analyte distribution is observed for samples prepared with the formation of microcrystals. The possible correlation between the ion signal variations observed in MALDI and the uniformity of the analyte distribution obtained by the confocal microscopic imaging method is discussed. Finally, a double-imaging method involving the use of two analytes with different labeling groups is demonstrated. It is found that different analytes are not coherently distributed in the matrix crystals.     

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.     

Digital microfluidics with in-line sample purification for proteomics analyses with MALDI-MS

An in-line sample purification method for MALDI-MS, which relies on the electrowetting-on-dielectric (EWOD)-based technique for digital microfluidics, is reported. In this method, a droplet containing peptides and impurities is moved by EWOD and deposited onto a Teflon-AF surface. A droplet of water is subsequently moved over the spot, where it dissolves and removes the impurities. A droplet containing MALDI matrix is then moved to the spot, which is analyzed by MALDI-MS. This purification method reduces the number of salt adduct peaks caused by low concentrations of impurities (e.g., 20 mM sodium phosphate), and reduces or eliminates the catastrophic effects of high concentrations of impurities (e.g., 8 M urea). The method was used to purify spots made by depositing multiple droplets of contaminated peptides. Spectra from the purified spots showed an increase in the S/N ratio as a function of the number of droplets deposited; when not purified, the S/N ratio remained constant regardless of the number of droplets. Finally, the method was used to purify protein digests for peptide mass fragment (PMF) searches, and was shown to be more efficient than the conventional method of purification with reversed-phase-packed pipet tips. We anticipate this new, in-line sample purification technique for EWOD-MALDI-MS will enable development of integrated high-throughput proteomics analysis methodologies.     

Device for the reversed-phase separation and on-target deposition of peptides incorporating a hydrophobic sample barrier for matrix-assisted laser desorption/ionization mass spectrometry

The separation of peptide mixtures from proteolytic cleavage is often necessary prior to mass spectrometry (MS) to enhance sensitivity and peptide mapping coverage. When buffers, salts, and other higher abundance peptides/contaminants are present, competition for charge during the electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) processes can lead to ion suppression for the targeted analyte(s). In this note, a simple reversed-phase microcolumn sample separation and deposition device (Sep-Dep) is described. The use of this device improves or renders possible the analysis of complex or contaminated peptide mixtures by MALDI-MS. The method is simple and inexpensive and utilizes single-use low-cost Geloader-type columns packed with reversed-phase material. The device described utilizes an open column, allowing for a gradient or narrow-step gradient to be applied by any solvent delivery system or manually with a pipet. A key feature of the device is a deposition chamber that can be custom-built to hold any MALDI target. The Sep-Dep device is attached directly to an in-house vacuum line and draws solvent from the open-ended LC column. The elution of separated peptides is performed directly onto a target that has been treated with a hydrophobic barrier. This barrier effectively isolates fractions and improves the quality and morphology of the matrix crystals. The method produces efficient separations of proteolytic peptides, significantly reducing signal suppression effects in MALDI.     

Stretched tissue mounting for MALDI mass spectrometry imaging

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) combines information-rich chemical detection with spatial localization of analytes. For a given instrumental platform and analyte class, the data acquired can represent a compromise between analyte extraction and spatial information. Here, we introduce an improvement to the spatial resolution achievable with MALDI MSI conducted with standard mass spectrometric systems that also reduces analyte migration during matrix application. Tissue is placed directly on a stretchable membrane that, when stretched, fragments the tissue into micrometer-sized pieces. Scanning electron microscopy analysis shows that this process produces fairly homogeneous distributions of small tissue fragments separated and surrounded by areas of hydrophobic membrane surface. MALDI matrix is then applied by either a robotic microspotter or an artist's airbrush. Rat spinal cord samples imaged with an instrumental resolution of 50-250 μm demonstrate lipid distributions with a 5-fold high spatial resolution (a 25-fold increase in pixel density) after stretching compared to tissues that were not stretched.     

Electrochemically controlled solid-phase microextraction based on conductive polypyrrole films

Solid-phase microextraction (SPME) fiber coatings based on conductive polypyrrole films were prepared for the electrochemical extraction and desorption of ionic analytes. Simple preparation of each of the PPY extraction coatings on a platinum wire was possible with a constant potential method, but more importantly, cycling of the film between oxidation and reduction potentials facilitated the extraction and desorption of ionic analytes. The analytes were desorbed into a sample aliquot of water and were determined by flow injection analysis using a mass spectrometer. The fiber coatings and the developed electrochemical SPME method were found to be stable and reproducible (RSD < 5%; N = 5) and could be extended to several cations and anions, confirming the versatility of the approach. Preconcentration of the analyte on the fiber was also possible by repeating the processes to increase the amount of analyte extracted.     

Selective sampling of phosphopeptides for detection by MALDI mass spectrometry

The analysis of phosphopeptides by mass spectrometry (MS) is one of the most challenging tasks in proteomics. This is due to the lower isoelectric point (pI) of phosphopeptides, which leads to inefficient sample ionization in MS, particularly when competing with other peptides. The problem is compounded by the typical low abundance of phosphopeptides in biological samples. We describe here a simple nonsorptive method to isolate phosphopeptides based on their pI. A voltage is applied to selectively migrate the phosphopeptides into a capillary, which are negatively charged at acidic pH. The selectively sampled fraction is directly deposited onto MALDI sample target in nanoliter volumes (7-35 nL) for highly sensitive MS detection. No significant sample loss is evident in this procedure; hence, the MS was able to detect the isolated phosphopeptides at trace quantity. In this case, attomole-level detection limit is achieved for synthetic phosphopeptides (nM concentration and nL volume), from a mixture containing other peptides at up to 1 million times higher in concentration. Selective sampling was also applied to the tryptic digest of beta- and alpha-caseins to reveal the multiple phosphorylated peptides at the low-femtomole level using MALDI MS. Knowledge of pI based on the rejection/injection of peptides was found to be useful in peak assignment. To confirm the sequence of the selectively sampled peptides, fraction collection was performed for offline ESI MS/MS analysis.     

ATR-FT-IR membrane-based sensor for integrated microliquid-liquid extraction and detection

A novel straightforward membrane-based sensor, which uses attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy has been developed. The flow cell designed permits the on-line microliquid-liquid extraction of the target analyte into a organic solvent layer (OSL), which was deposited on the ATR surface using a sequential injection manifold. The aqueous and organic phases are separated via a commercial hydrophobic membrane placed on the PTFE piece of the cell. The main advantage of the proposed device is that the OSL can be created and regenerated in a continuous manner using the automatic manifold without opening the cell. The analytes are enriched into the OSL after diffusion through the membrane, which excludes the typical absorption bands of water. In addition, the behavior of different organic solvents was evaluated in order to increase the applicability and versatility of the proposed system. Finally, the analytical performance of the design was established for the detection and quantitation of Triton X100 in water.     

Oil-assisted sample preparation: a simple method for analysis of solid samples using matrix-assisted laser desorption/ionization mass spectrometry

Common mass spectrometric techniques, e.g., electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), require samples to be soluble in suitable solvents. Samples with solubility problems have difficulties for their mass spectrometric characterization. In this paper, an oil-assisted sample preparation (OASP) method was introduced for the analysis of solid samples using MALDI-MS. The novel method involves the use of a droplet of oil (i.e., paraffin oil) as the mixing and loading media for solid analyte and solid matrix. Using this method, rapid on-target sample preparation can be easily achieved, and only a transferable minimal amount of analyte and matrix is required. This method was demonstrated to be applicable for a wide range of analytes, including poorly soluble organic compounds, polymers, organometallic compounds, membrane peptides, and biological solid samples. The novel method can also be used for the analysis of "wet" and solution samples. The limit of detection of the OASP MALDI-MS was determined to be 1 ng with reserpine.     

Sample preparation for MALDI mass spectrometry using an elastomeric device reversibly sealed on the MALDI target

A new method for improving low-concentration sample recovery and reducing sample preparation steps in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is presented. In the conventional approach, samples are typically desalted and/or concentrated with various techniques and deposited on the MALDI target as small droplets. In this work, we describe a new approach in which an elastomeric device is reversibly sealed on the MALDI target to form a multi-well plate with the MALDI target as the base of the plate. The new format allows a larger volume (5-200 microL) of samples to be deposited on each spot and a series of sample handling processes, including desalting and concentrating, to be performed directly on the MALDI target. Several advantages have been observed: (i) multiple sample transferring steps are avoided; (ii) recovery of low-concentration peptides during sample preparation is improved using a novel desalting method that utilizes the hydrophobic surface of the elastomeric device; and (iii) sequence coverage of the peptide mass fingerprinting map is improved using a novel method in which proteins are immobilized on the hydrophobic surface of the elastomeric device for in-well trypsin digestion, followed by desalting and concentrating the digestion products in the same well.     

Noncovalent antibody immobilization on porous silicon combined with miniaturized solid-phase extraction (SPE) for array based immunoMALDI assays

This paper presents a new strategy to combine the power of antibody based capturing of target species in complex samples with the benefits of microfluidic reverse phase sample preparation on an integrated sample enrichment target (RP-ISET) and the analysis speed of MALDI MS. The immunoaffinity step is performed on an in-house developed 3D-structured high surface area porous silicon (PSi) matrix, which allows efficient antibody immobilization by surface adsorption without any coupling agents in 30-60 min. The hydrophilic nature of the porous silicon surface at the molecular level displays a low adsorption of background peptides when exposed to complex digests or plasma samples, improving the conditions for the antigen specific extraction and subsequent readout. At the same time, the hydrophobic behavior, due to the nanostructured surface, of the PSi material facilitates liquid confinement during the assay. Using a footprint conforming to the standard for 384 well microplates, direct adaption of the protocol into standard sample handling robots is possible. The performance of the proposed immunoaffinity PSi-ISET immunoMALDI (iMALDI) assay was evaluated by specific detection of angiotensin I at a 10 femtomol level in diluted plasma samples (10 μL, 1 nM).