Subpicomole Level Research Articles

Acridine-based dyes as high-performance near-infrared Raman reporter molecules for cell imaging.

A surface-enhanced Raman scattering (SERS) nanoprobe has been proven to be a promising tool for near-infrared (NIR) biomedical imaging and diagnosis because of its high sensitivity and selectivity. However, the development of NIR SERS reporters has been a bottleneck impeding the preparation of ultrasensitive SERS probes. Herein, we report the design and synthesis of a series of SERS reporters in the NIR region based on 10-methylacridine (AD). The AD nanotags (gold nanostar–AD molecules–BSA, AuNS–AD–BSA) exhibit appreciable SERS signals and can be detected at as low as the sub-picomole level. The results of in vitro imaging experiments show that it can be used in live-cell delineation.

Real-time laser induced chemical derivatizations of peptide N-Terminus for in-situ mass spectrometric sequencing at sub-picomole and nanosecond scale

Distinguishing b- and y-ions is essential to compute amino acid sequences from either N- or C-terminus in mass spectrometry. We described herein a solvent free and real time on-plate derivatization approach that can tag N-terminus of peptides at microliter level with p-chlorobenzaldehyde or 2-hydroxy-5-methylisophthalaldehyde for matrix assisted laser desorption ionization mass spectrometry (MALDI MS). Less than 1 μL of sample solutions can be directly mixed with equal volumes of p-chlorobenzaldehyde or 2-hydroxy-5-methylisophthalaldehyde and α-cyano-4-hydroxycinnamic acid (CHCA), a matrix compound to co-crystalize with analytes for efficient absorption of laser energy and peptide ionization. When the mixture spotted on the sample plate is irradiated with the 3rd harmonic (355 nm) of Nd3+:YAG laser pulses (3 ns width), N-terminal amine groups of peptides instantly react with carbonyl groups of chlorobenzaldehyde or 2-hydroxy-5-methylisophthalaldehyde. Resultant peptides carrying with on-plate formed azomethine group (-CN-) are simultaneously protonated and isolated as precursor ions for subsequent collision-activated dissociation. The mass shift with unique Cl isotopic signature unambiguously distinguishes b ions from y ions and other ions. This method does not need extensive sample preparation and is useful for those samples with limited quantities down to sub-picomole level in sub-microliter volumes. The efficiency was demonstrated with synthetic peptides and tryptic peptides of model proteins. It was found that 2-hydroxy-5-methylisophthalaldehyde provides improved yield for peptides containing lysine residues. Unknown proteins of human saliva and bovine milk as well as phosphopeptides have been identified.

Sub-picomole level photoelectrochemical sensing of l-cysteine based on plasmonic silver nanoparticles modified hierarchically structured zinc oxide

A novel photoelectrochemical (PEC) sensor for l-cysteine was fabricated using in situ plasmonic silver nanoparticles modified hierarchically structured zinc oxide. The fabricated PEC sensor was based on Ag–S coordination chemistry and the collective oscillatory motion of electrons resulting from the coherent excitation of the “free” electrons in the conduction band of silver nanoparticles. The energetic electrons from the surface plasmon of the silver nanoparticles may be injected into the conduction band of the hierarchically structured zinc oxide and then fast transferred to the ITO electrode. However, the enhanced photocurrent produced by the plasmonic silver nanoparticles can be efficiently inhibited due to the bonding of Ag–S upon the simulated sunlight irradiation and at the bias voltage of 0.2 V. Under the optimized condition, the fabricated PEC sensor was linear with the concentration of l-cysteine in the range from 0.67 to 34.77 pmol L−1 with the detection limit of 0.21 pmol L−1. The presence of definite concentration of conventional species did not interfere in the detection of l-cysteine and the results agreed well with those obtained by HPLC. This novel plasmonic PEC sensor has good performances in high sensitivity, good selectivity, easy operation, economy, short detection time, and portability. The strategy of localized surface plasmon resonance by in situ generation of silver nanoparticles provides an alternative way to construct a PEC sensor of l-cysteine.

Sensitive Analyses of Neutral N-Glycans using Anion-Doped Liquid Matrix G3CA by Negative-Ion Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Negative-ion fragmentation of N-glycans has been proven to be more informative than that of positive-ion. In particular, it defines structural features such as the specific composition of the two antennae and the location of fucose. However, negative-ion formation of neutral N-glycans by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) remains a challenging task, and the detection limit of N-glycans in negative-ion mode is merely at the subpicomole level. Thus, practical applications are limited. In this study, combinations of five liquid matrices and nine anions were used to ionize N-glycans as anionic adducts, and their performances for sensitive analyses were evaluated. The best results were obtained with anion-doped liquid matrix G(3)CA, which consists of p-coumaric acid and 1,1,3,3-tetramethylguanidine; the detection limits of anion adducted N-glycans were 1 fmol/well for NO(3)(-), and 100 amol/well for BF(4)(-). Negative-ion MS(2) spectra of 1 fmol N-glycans were successfully acquired with a sufficient signal-to-noise ratio and were quite useful for MS-based structural determination. The anion-doped G(3)CA matrix opens the way for sensitive and rapid analysis of neutral N-glycans in negative-ion MALDI at a low femtomole level.

Amine-Functionalized Sol–Gel-Based Lab-in-a-Pipet-Tip Approach for the Fast Enrichment and Specific Purification of Phosphopeptides in MALDI-MS Applications

Amine-functionalized sol-gels were investigated for the enrichment and purification of phosphopeptides from digested protein mixture solutions. Tetramethylorthosilicate (TMOS) and N'[3-(trimethoxysilyl)-propyl]-diethylenetriamine (TPDA) were used in a 1:1 mole ratio in the production of amine-functionalized sol-gels. The sol-gel network was then used for phosphopeptide enrichment. Phosphopeptide enrichment onto the synthesized amine-functionalized sol-gels was performed using an enolase digested peptide mixture, a β-casein digested peptide mixture, as well as these digested peptide mixtures contaminated 50-fold with bovine serum albumin (BSA). Moreover, phosphopeptide enrichment was successfully performed using nonfat milk as a highly contaminated and complex material. In each phosphopeptide enrichment and purification process, only phosphopeptides were enriched and separated from the other digested peptides. Phosphopeptides were adsorbed onto the amine-functionalized sol-gels at pH 4.0 and eluted at pH 1.0 using trifluoroacetic acid (TFA). For phosphopeptide analysis by MALDI-MS, a 2,5-dihydroxybenzoic acid matrix containing 1.0% phosphoric acid was used to overcome the degradation of phosphopeptides and provide high intensity phosphopeptide protonated molecular ion signal intensities. It was also found that phosphopeptide detection limits were improved to approximately 10 femtomoles. For rapid and specific phosphopeptide enrichment and purification, sol-gel materials were placed in a 10 μL pipet tip with glass wool on either side. Phosphopeptide enrichment from digested peptide mixtures was performed in a very short time (less than 1 min) at subpicomole levels using this novel lab-in-a-pipet-tip approach.

Isothermal Discrimination of Single-Nucleotide Polymorphisms via Real-Time Kinetic Desorption and Label-Free Detection of DNA Using Silicon Photonic Microring Resonator Arrays

We report a sensitive, label-free method for detecting single-stranded DNA and discriminating between single nucleotide polymorphisms (SNPs) using arrays of silicon photonic microring resonators. In only a 10 min assay, DNA is detected at subpicomole levels with a dynamic range of 3 orders of magnitude. Following quantitation, sequence discrimination with single nucleotide resolution is achieved isothermally by monitoring the dissociation kinetics of the duplex in real-time using an array of SNP-specific capture probes. By leveraging the capabilities of the microring resonator platform, we successfully generate multiplexed arrays to quickly screen for the presence and identity of SNPs and show the robustness of this methodology by analyzing multiple target sequences of varying GC content. Furthermore, we show that this technique can be used to distinguish both homozygote and heterozygote alleles.

Label free sub-picomole level DNA detection with Ag nanoparticle decorated Au nanotip arrays as surface enhanced Raman spectroscopy platform

Label free optical sensing of adenine and thymine oligonucleotides has been achieved at the sub-picomole level using self assembled silver nanoparticles (AgNPs) decorated gold nanotip (AuNT) arrays. The platform consisting of the AuNTs not only aids in efficient bio-immobilization, but also packs AgNPs in a three dimensional high surface area workspace, assisting in surface enhanced Raman scattering (SERS). The use of sub-10 nm AgNPs with optimum inter-particle distance ensures amplification of the chemically specific Raman signals of the adsorbed adenine, thymine, cytosine and guanine molecules in SERS experiments. High temporal stability of the Raman signals ensured reliable and repeatable DNA detection even after three weeks of ambient desk-top conservation. This facile architecture, being three dimensional and non-lithographic, differs from conventional SERS platforms.

Sodium dodecyl sulfate sensitized electrochemical method for sub-picomole level determination of topotecan hydrochloride at a novel disposable electrode

A sub-picomole level topotecan hydrochloride determination method was first proposed using sodium dodecyl sulfate (SDS) as a sensitized reagent at a novel disposable electrode (an improved wax-impregnated graphite electrode). The effects of different kinds of surfactants on the electrochemical response to topotecan hydrochloride were examined. The results indicate the electrochemical signal was apparently improved by SDS. At optimal conditions, the oxidative peak current increased linearly with the logarithm of concentrations for topotecan hydrochloride in the ranges of 2.0 × 10−12 to 1.0 × 10−11 mol/L and 8.0 × 10−11 to 8.0 × 10−10 mol/L with a detection limit of 6.4 × 10−13 mol/L. The proposed method could be applied in determining topotecan hydrochloride in urine.

Glycan reductive isotope labeling for quantitative glycomics

Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed glycan reductive isotope labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [ 12C 6]aniline and [ 13C 6]aniline. These dual-labeled aniline-tagged glycans can be recovered by reverse-phase chromatography and can be quantified based on ultraviolet (UV) absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins, using this method. This technique allows linear relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of glycomics.

Focused Differential Glycan Analysis with the Platform Antibody-assisted Lectin Profiling for Glycan-related Biomarker Verification

Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery.

Assaying Small-Molecule−Receptor Interactions by Continuous Flow Competitive Displacement Chromatography/Mass Spectrometry

Small-molecule screening techniques that employ mass spectrometry detection have been highly successful. However, the inability of conventional techniques, such as frontal affinity chromatography-mass spectrometry (FAC-MS), to easily identify weak binding molecules (i.e., Kd >or= 1 microM) using small amounts of target protein (subpicomole levels) represents a significant impediment to the widespread use of the method in the routine screening of low-abundance membrane receptors. This limitation is particularly notable in the early stages of the drug discovery process, as weak binding molecules can serve as useful leads for targets with no known ligand or when existing tight binding ligands have little therapeutic value. Competitive assay methods involving the displacement of an indicator ligand offer a more sensitive alternative, as the ability to generate an appreciable signal through various methods, including transient overconcentrations of indicator compounds, provides an unambiguous means for identifying weak affinity ligands. In this work we describe a continuous flow competitive assay based on the principles of FAC-MS that can be widely used to identify and characterize weak affinity ligands using low levels of the nicotinic acetylcholine receptor from Torpedo californica (nAChR). The validity of the assay is shown through the ability to identify nicotine (Kd approximately 1 microM) with columns containing <2 pmol of binding sites. Multiple injections of nicotine on a single column produce reproducible peaks in the signal of the indicator compound, epibatidine (Kd approximately 2 nM) showing minimal degradation in signal intensity between trials. The intensity of the peaks is dependent on the concentration of nicotine being injected, and binding curves can be generated through multiple injections on the same column. We investigate and optimize various parameters, including assay speed and concentrations, and demonstrate an automated assay format with the potential for use as a high-throughput screening tool. The ability to screen for weak binders of more pharmacologically relevant membrane receptors in a high-throughput screening format is discussed.

Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters

A new approach to determine in vivo pools of coenzyme A (CoA) and short chain acyl-CoA thioesters is reported. The metabolites released by extraction with trichloroacetic acid are recovered and quantitatively dephosphorylated by treatment with shrimp alkaline phosphatase. Following phosphatase removal, the dephosphorylated CoA metabolites are quantitatively rephosphorylated by treatment with [γ- 33P]ATP plus a dephospho-CoA kinase. The resulting radioactive CoA metabolites are then separated by reverse-phase high-performance liquid chromatography and quantitated by scintillation counting. Due to the enzymatic radiophosphorylation, the assay is specific for CoA and its short chain thioesters and is sensitive to sub-picomole levels of these compounds.

Genotoxicity Screening Using Biocatalyst/DNA Films and Capillary LC−MS/MS

Detection of DNA adducts can serve as a basis for genotoxicty screening of new chemicals and drugs. We report here a simple, sensitive procedure for this purpose using films containing DNA and a biocatalyst to mimic the metabolic action of human liver cytochrome P450s. DNA adducts formed from an in-situ-generated toxic metabolite (styrene oxide) were detected at subpicomole levels after neutral thermal hydrolysis of the DNA films and analysis with capillary liquid chromatography with on-line column preconcentration and MS/MS detection. An on-line column switching system allowed for increased sample loading volume and analyte preconcentration. This approach provides an estimate of the relative rate of DNA damage.

Phosphoproteomics by mass spectrometry and classical protein chemistry approaches

The general fields of biological sciences have seen phenomenal transformations in the past two decades at the level of data acquisition, understanding biological processes, and technological developments. Those advances have been made partly because of the advent of molecular biology techniques (which led to genomics) coupled to the advances made in mass spectrometry (MS) to provide the current capabilities and developments in proteomics. However, our current knowledge that approximately 30,000 human genes may code for up to 1 million or more proteins disengage the interface between the genome sequence database algorithms and MS to generate a major interest in independent de novo MS/MS sequence determination. Significant progress has been made in this area through procedures to covalently modify peptide N- and C-terminal amino-acids by sulfonation and guanidination to permit rapid de novo sequence determination by MS/MS analysis. A number of strategies that have been developed to perform qualitative and quantitative proteomics range from 2D-gel electrophoresis, affinity tag reagents, and stable-isotope labeling. Those procedures, combined with MS/MS peptide sequence analysis at the subpicomole level, permit the rapid and effective identification and quantification of a large number of proteins within a given biological sample. The identification of proteins per se, however, is not always sufficient to interpret biological function because many of the naturally occurring proteins are post-translationally modified. One such modification is protein phosphorylation, which regulates a large array of cellular biochemical pathways of the biological system. Traditionally, the study of phosphoprotein structure-function relationships involved classical protein chemistry approaches that required protein purification, peptide mapping, and the identification of the phosphorylated peptide regions and sites by N-terminal sequence analysis. Recent advances made in mass spectrometry have clearly revolutionized the studies of phosphoprotein biochemistry, and include the development of specific strategies to preferentially enrich phosphoproteins by covalent-modifications that incorporate affinity tags that use the physicochemical properties of phosphoaminoacids. The phosphoserine/phosphothreonine-containing proteins/peptides are derivatized under base-catalyzed conditions by thiol agents; mono- and di-thiol reagents both have been used in such studies. The thiol agent may have: (i) an affinity tag for protein enrichment; (ii) stable-isotopic variants for relative quantitation; or (iii) a combination of the moieties in (i) and (ii). These strategies and techniques, together with others, are reviewed, including their practical application to the study of phosphoprotein biochemistry and structure-function. The consensus of how classical protein chemistry and current MS technology overlap into special case of proteomics, namely "phosphoproteomics," will be discussed.

Glycopeptide analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry reveals novel features of horseradish peroxidase glycosylation.

We explored matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF/TOF) mass spectrometry for the analysis of N-glycosylated peptides, using horseradish peroxidase (HRP) as a test case. Two different types of cleavage were observed in the TOF/TOF fragmentation spectra: Firstly, cleavages of peptide bonds yielded fragments with the attached N-glycans staying intact, thus revealing information on peptide sequence and glycan attachment site. Secondly, fragmentation of the glycan moiety was characterized by cleavage of glycosidic bonds as well as a (0,2)X-ring fragmentation of the innermost N-acetylglucosamine of the chitobiose core. Loss of the complete N-glycan moiety occurred by cleavage of both the N-glycosidic bond and the side-chain amide group of the N-glycosylated asparagine, yielding a characteristic peak doublet with a mass difference of 17 Da, which revealed the individual masses of the N-glycan and the peptide moiety. Analysis of a HRP tryptic digest at the sub-picomole level allowed the characterization of various N-glycosylated peptides including those with internal disulfide linkages, a glycopeptide linked via a disulfide bond to another peptide, and a 5 kDa glycopeptide carrying two N-glycans. The potential of our approach was illustrated by the detection of the following novel features of HRP glycosylation: (i) The conjugation of a xylosylated trimannosyl N-glycan without core-fucosylation to site Asn316, showing for the first time unambiguously the occupation of this site; and (ii) A disaccharide N-acetylhexosamine1deoxyhexose1 attached to N-glycosylation sites Asn285 and Asn298, which might represent a Fuc(alpha1-3)GlcNAc- moiety arising from the processing of N-glycans by a horse-radish endoglycosidase during biosynthesis of HRP.

Virtual two-dimensional gel electrophoresis of high-density lipoproteins.

High-density lipoproteins (HDLs) isolated by immunoaffinity chromatography and separated by immobilized pH gradient-isoelectric focusing (IPG-IEF) were examined by mass spectrometry directly, applying a new proteomics technology, virtual two-dimensional (2-D) gel electrophoresis. A preliminary examination of HDL particles has revealed at least 42 unique masses for protein species with isoelectric points between pH 5.47-5.04, some of which have not been observed previously. By delivering masses of intact proteins from complex cellular mixtures in a format that correlates directly to classical 2-D gel analyses, virtual 2-D gel electrophoresis constitutes a general discovery tool to expose and monitor protein isoforms and post-translational modifications. Furthermore, its general ability to deliver ions from sub-picomole level proteins enmeshed in complex cellular mixtures potentially fulfills the need of top-down proteomics to obtain intact protein ions from microscale samples. Additional comparison of such data to 2-D gel analyses and their identified proteins may elucidate the functions of the individual apolipoprotein components and the cardioprotective effects of HDL.

Mass spectrometric characterization of two novel inflammatory peptides from the venom of the social wasp Polybia paulista.

The social wasp P. paulista is relatively common in southeast Brazil causing many medically important stinging incidents. The seriousness of these incidents is dependent on the amount of venom inoculated by the wasps into the victims, and the characteristic envenomation symptoms are strongly dependent on the types of peptides present in the venom. In order to identify some of these naturally occurring peptides available in very low amounts, an analytical protocol was developed that uses a combination of reversed-phase and normal-phase high-performance liquid chromatography (HPLC) of wasp venom for peptide purification, with matrix-assisted laser desorption/ionization time-of-flight post-source decay mass spectrometry (MALDI-Tof-PSD-MS) and low-energy collision-induced dissociation (CID) in a quadrupole time-of-flight tandem mass spectrometry (QTof-MS/MS) instrument for peptide sequencing at the sub-picomole level. The distinction between Leu and Ile was achieved both by observing d-type fragment ions obtained under CID conditions and by comparison of retention times of the natural peptides and their synthetic counterparts (with different combinations of I and/or L at N- and C-terminal positions). To distinguish the isobaric residues K and Q, acetylation of peptides was followed by Q-Tof-MS analysis. The primary sequences obtained were INWLKLGKMVIDAL-NH(2) (MW 1611.98 Da) and IDWLKLGKMVMDVL-NH(2) (MW 1658.98 Da). Micro-scale bioassay protocols characterized both peptides as presenting potent hemolytic action, mast cell degranulation, and chemotaxis of polymorphonucleated leukocyte (PMNL) cells. The primary sequences and the bioassay results suggest that these toxins constitute members of a new sub-class of mastoparan toxins, directly involved in the occurrence of inflammatory processes after wasp stinging.

Sub-picomole colorimetric single nucleotide polymorphism discrimination using oligonucleotide-nanoparticle conjugates.

Oligonucleotide-gold nanoparticle conjugates are employed to demonstrate selective colorimetric discrimination of a cystic fibrosis-related genetic mutation in a model oligonucleotide system. Specifically, three strand oligonucleotide complexes are employed, wherein two probe oligonucleotide-gold nanoparticle conjugates are linked together by a third target oligonucleotide strand bearing the chosen CF-related mutation. By monitoring the solution optical absorption behaviour of the complexes as a function of temperature, melting profiles may be accurately acquired and reproducibly compared. Following this method, fully complementary sequences are successfully distinguished from mismatched sequences, with single base mismatch resolution, for the V232D mutation. To extend the detection sensitivity of this oligonucleotide-nanoparticle conjugate assay, a novel and compact LED-based optical monitoring system capable of sub-picomole level colorimetric SNP discrimination is also demonstrated.

Improved beta-elimination-based affinity purification strategy for enrichment of phosphopeptides.

Alkaline-induced beta-elimination of phosphate from phosphoserine and phosphothreonine residues followed by addition of an affinity tag has recently been pursued as a strategy for enriching phosphorylated species from complex mixtures. Here we report the use of an introduced thiol tag as the ligand for affinity purification via disulfide exchange with an activated thiol resin and the development of a protocol to improve the sensitivity considerably over previous reports (i.e., to subpicomole levels.) During our experiments, we observed a side reaction in which water was eliminated from unmodified serine residues. This side reaction resulted in the introduction of the affinity tag into unphosphorylated proteins, confounding attempts to specifically purify phosphoproteins from mixtures. Unchecked, this side reaction will also prevent application of the beta-elimination strategy to phosphopeptide samples where the phosphorylated species are minor components (i.e., most current phosphoproteomics applications). Quantitation of the side reaction products using three synthetic unphosphorylated peptides showed varying conversion efficiencies; at maximum, 1.7% of unphosphorylated peptide was converted to the affinity-tagged form. Inclusion of EDTA into the reaction reduced the side reaction but also greatly reduced the conversion efficiency of one of the phosphoserine residues of ovalbumin, suggesting a role for trace metal ions in the beta-elimination chemistry. Despite the presence of the side reaction, the affinity strategy was shown to be effective at enriching phosphopeptides from fairly complex peptide mixtures. The strategy was applied to the analysis of in vitro phosphorylation of bovine synapsin I by Ca(2+)/calmodulin-dependent kinase II, resulting in the identification of four phosphorylation sites, two of which have not been previously reported.

A microfluidic electrocapture device in sample preparation for protein analysis by MALDI mass spectrometry.

The design and operation of a microfluidic device for sample preparation in MALDI mass spectrometry of peptides and proteins is described. It is particularly useful for proteomics applications and for mass determination of proteins in salt- and detergent-containing solutions. The system consists of a flow channel with two conductive areas or electrical junctions where proteins and peptides are retained by means of an electric field. The microfluidic device is made of PEEK tubing, and the junctions are covered with a conductive polymeric membrane. A syringe pump connected to the device produces a flow stream, and injection of sample is carried out manually via hydrodynamic pressure. Proteolytic peptides and intact proteins in salt- and detergent-containing acidic media were captured at the cathode junction followed by exchange of the original solution to a solvent suitable for subsequent mass spectrometry. Using this principle, a significant desalting effect was obtained for tryptic peptides in mass-mapping experiments. Protein sequence coverages were high (up to 40%) at subpicomole levels with results better than those obtained using reversed-phase solid-phase extraction. In contrast to the latter technique, the microfluidic device has the capacity to efficiently remove detergents such as CHAPS before peptide mapping and protein analysis.