基于整体材料的微流控芯片反相液相色谱-串联质谱平台用于蛋白质分析

微流控芯片高效液相色谱-串联质谱系统具有高通量、高灵敏度等优点,已成为生物样品分析的热点领域之一。本文在玻璃芯片上以甲基丙烯酸十二酯(LMA)和三羟甲基丙烷三甲基丙烯酸酯(TMPTMA)为单体,制备了以聚丙烯酸酯整体材料为固定相的捕集柱和分离柱。通过在芯片通道末端连接细内径的毛细管作为芯片-质谱接口,并以常规的液相色谱泵和微阀控制流体,构建了芯片反相液相色谱-电喷雾串联质谱(RPLC-ESI-MS/MS)平台,并将其用于分析牛血清白蛋白(BSA)的酶解产物。经过3次平行分析,BSA的序列覆盖率分别为39.37%、37.89%和34.10%(相对标准偏差为7.3%)。采用不同批次制作的芯片构建RPLC-ESI-MS/MS平台,对BSA酶解产物进行分析,其序列覆盖率相当。上述结果表明,该平台具有灵敏度高和重现性好等优点,有望用于蛋白质样品的快速分离和高灵敏度鉴定。     

Proteolysis in microfluidic droplets: an approach to interface protein separation and peptide mass spectrometry

A versatile microreactor protocol based on microfluidic droplets has been developed for on-line protein digestion. Proteins separated by liquid chromatography are fractionated in water-in-oil droplets and digested in sequence. The microfluidic reactor acts also as an electrospray ionization emitter for mass spectrometry analysis of the peptides produced in the individual droplets. Each droplet is an enzymatic micro-reaction unit with efficient proteolysis due to rapid mixing, enhanced mass transfer and automated handling. This droplet approach eliminates sample loss, cross-contamination, non-specific absorption and memory effect. A protein mixture was successfully identified using the droplet-based micro-reactor as interface between reverse phase liquid chromatography and mass spectrometry.     

Low-molecular weight protein profiling of genetically modified maize using fast liquid chromatography electrospray ionization and time-of-flight mass spectrometry

In this work, the use of liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC-TOFMS) has been evaluated for the profiling of relatively low-molecular weight protein species in both genetically modified (GM) and non-GM maize. The proposed approach consisted of a straightforward sample fractionation with different water and ethanol-based buffer solutions followed by separation and detection of the protein species using liquid chromatography with a small particle size (1.8 μm) C(18) column and electrospray-time-of-flight mass spectrometry detection in the positive ionization mode. The fractionation of maize reference material containing different content of transgenic material (from 0 to 5% GM) led to five different fractions (albumins, globulins, zeins, zein-like glutelins, and glutelins), all of them containing different protein species (from 2 to 52 different species in each fraction). Some relevant differences in the quantity and types of protein species were observed in the different fractions of the reference material (with different GM contents) tested, thus revealing the potential use of the proposed approach for fast protein profiling and to detect tentative GMO markers in maize.     

Multidimensional nano-HPLC for analysis of protein complexes

The analysis of macromolecular protein complexes is an important factor in understanding most cellular processes, e.g., protein transport into cell organells, signal transduction via biological membranes, apoptosis, energy metabolism, directed motion of cells, and cell division. These complexes are not only built of various numbers of different proteins but also of prosthetic groups and RNA molecules. To understand the role each protein plays in a complex, a complete analysis of all protein compounds is necessary. Therefore, several separation steps have to be coupled to mass spectrometry to identify the proteins. In this work, we describe the application of multidimensional liquid chromatography, SCX-RP-LC as well as SAX-RP-LC, coupled to electrospray ion trap mass spectrometry. Tryptic digested ribosomes were separated by ion exchange chromatography manually collected and prepared for reversed phase chromatography to analyze the peptides via nano-ESI mass spectrometry. The total numbers of identified proteins are compared in consideration of the separation method (SCX-RP versus SAX-RP).     

Resolution and identification of the protein components of the photosystem II antenna system of higher plants by reversed-phase liquid chromatography with electrospray-mass spectrometric detection

Reversed-phase liquid chromatography (RPLC) was interfaced to mass spectrometry (MS) with an electrospray ion (ESI) source for the separation and accurate molecular mass determination of the individual intrinsic membrane proteins that comprise the photosystem II (PS II) major light-harvesting complex (LHC II) and minor (CP24, CP26 and CP29) antenna system, whose molecular masses range between 22,000 and 29,000. PS II is a supramolecular complex intrinsic of the thylacoid membrane, which plays the important role in photosynthesis of capturing solar energy, and transferring it to photochemical reaction centers where energy conversion occurs. The protein components of the PS II major and minor antenna systems were extracted from spinach thylacoid membranes and separated using a butyl-silica column eluted by an acetonitrile gradient in 0.05% (v/v) aqueous trifluoroacetic acid. On-line electrospray MS allowed accurate molecular mass determination and identification of the protein components of PS II major and minor antenna system. The proposed RPLC-ESI-MS method holds several advantages over sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the conventional technique for studying membrane proteins, including a better protein separation, mass accuracy, speed and efficiency.     

The novel use of gas chromatography-ion mobility-time of flight mass spectrometry with secondary electrospray ionization for complex mixture analysis

Increasing the dimensionality of an analysis enables more detailed and comprehensive investigations of complex mixtures. One dimensional separation techniques like gas chromatography (GC) and ion mobility spectrometry (IMS) provide limited chemical information about complex mixtures. The combination of GC, ion mobility spectrometry, and time-of-flight mass spectrometry (GC-IM-TOFMS) provides three-dimensional separation of complex mixtures. In this work, a hybrid GC-IM-TOFMS with a secondary electrospray ionization (SESI) source provided four types of analytical information: GC retention time, ion mobility drift time, mass-to-charge ratios, and ion intensity. The use of secondary electrospray ionization enables efficient and soft ionization of gaseous sample vapors at atmospheric pressure. Several complex mixtures, including lavender and peppermint essential oils, were analyzed by GC-SESI-IM-TOFMS. The resulting 3D data from these mixtures, each containing greater than 50 components, were plotted as 3D projections. In particular, post-processed data plotted in three dimensions showed that many mass selected GC peaks were resolved into different ion mobility peaks. This technique shows clear promise for further in-depth analyses of complex chemical and biological mixtures.     

Multidimensional system enabling deglycosylation of proteins using a capillary reactor with peptide-N-glycosidase F immobilized on a porous polymer monolith and hydrophilic interaction liquid chromatography–mass spectrometry of glycans

A reactor with immobilized peptide-N-glycosidase F on a monolithic polymer support in a capillary has been developed that allows fast and efficient release of N-linked glycans from immunoglobulin G molecules. Two different monolithic scaffolds based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) and poly(butyl methacrylate-co-ethylene dimethacrylate) were prepared. A multistep photografting process was used to reduce non-specific adsorption of proteins and to obtain support containing reactive azlactone functionalities enabling the preparation of highly active immobilized peptide-N-glycosidase F. Performance of these reactors was determined through glycan release from several glycoproteins including ribonuclease B, chicken albumin, and human immunoglobulin G and their detection by matrix-assisted laser desorption-ionization/time-of-flight mass spectrometry. The optimized reactor was integrated into a multidimensional system comprising on-line glycan release and their separation via hydrophilic interaction liquid chromatography followed by electrospray ionization/time-of-flight mass spectrometry detection. Using the optimized monolithic reactor with immobilized peptide-N-glycosidase F, human immunoglobulin G was deglycosylated at room temperature in 5.5 min to an extent similar to that achieved with soluble enzyme after 24 h at 37 °C.     

Preliminary investigation of the application of on-line membrane extraction of trifluoroacetic acid as an aid to improvement of negative ion electrospray mass spectrometry data

We have recently investigated the biodegradation of a number of acidic aromatic compounds that give excellent chromatography using trifluoroacetic acid (TFA) based HPLC methods. Unfortunately HPLC methods using TFA are not usually compatible with detection by negative ion mass spectrometry as TFA suppresses ionisation of the analyte during the electrospray process. We present a preliminary investigation of the use of an anion-exchange micro-membrane suppressor to remove TFA on-line post column with the aim of improvement of mass spectral data using an aromatic acid as an example, Thus LC-MS using a TFA based HPLC method with negative ion mass spectral detection is shown to be possible with good sensitivity.     

Formation of iron complexes from trifluoroacetic acid based liquid chromatography mobile phases as interference ions in liquid chromatography/electrospray ionization mass spectrometric analysis

Two unexpected singly charged ions at m/z 1103 and 944 have been observed in mass spectra obtained from electrospray ionization mass spectrometric analysis of liquid chromatography effluents with mobile phases containing trifluoroacetic acid (TFA) that severely interfered with sample analysis. Accurate mass measurement and tandem mass spectrometry studies revealed that these two ions are composed of three components; clusters of trifluoroacetic acid, clusters of mass 159 and iron. Formation of these ions is inhibited by removing TFA from the mobile phases and using formic acid in its place, replacing the stainless steel union with a titanium union or by adding a small blank fused-silica capillary column between the chromatography column and the electrospray tip via a stainless steel union without any adverse effects to chromatographic separation, peak broadening or peptide identifications.     

Linking protein fractionation with multidimensional monolithic reversed-phase peptide chromatography/mass spectrometry enhances protein identification from complex mixtures even in the presence of abundant proteins

Recently, multidimensional shotgun proteomics has proven to be an alternative technology able to identify hundreds of proteins from single samples. Two major limitations of the technology are the presence of high abundance proteins (e.g. RUBISCO in plant leaf tissue) and the enormous number of co-eluting peptides that overstrain the loading and resolving capacity of conventional particle-packed columns as well as the capacity of electrospray ionisation due to ion suppression. Here, the coupling of fast performance liquid chromatography (FPLC) pre-fractionation of an Arabidopsis leaf protein extract and subsequent two-dimensional liquid chromatography/mass spectrometry with improved resolution using a monolithic silica C18 capillary column allowed the identification of 1032 unique proteins in a single 4 mg total protein plant leaf tissue sample. The reassignment of peptide IDs to distinct FPLC protein fractions enhances the identification procedure, especially in the case of present protein isoforms. The proposed strategy is useful to detect proteins otherwise not seen in conventional multidimensional chromatography/mass spectrometry approaches.     

Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification

A peptide separation strategy that combines two-dimensional (2-D) liquid chromatography (LC)-capillary zone electrophoresis (CZE) with tandem mass spectrometry (MS/MS) is described for the identification of proteins in complex mixtures. To test the effectiveness of this strategy, a serum sample was depleted of the high-abundance proteins by methanol precipitation, digested with trypsin to generate a complex peptide mixture, and separated into 96 fractions by reversed-phase (RP)-LC. Compared to ion-exchange LC separations, RPLC provides much higher resolution and peak capacity. Fractions were collected off-line from the RPLC separation, and subjected to short 20 min CZE separations. The separated zones were introduced to the mass spectrometer through a sheathless electrospray ionization interface that is integrated on the separation capillary. The ease of fabrication of the interface and its durability allowed for the analysis of all fractions on a single capillary in a relatively short analysis time. A stable electrospray was produced at nanoliter flowrates by augmenting analyte electrophoretic and electroosmotic mobilities with pressure-assisted flow. Unlike first-dimensional ion-exchange LC fractionation, where there is a large degree of overlap, the CZE-MS results show less than 15% overlap between neighboring RPLC fractions.     

Operational variables in high-performance liquid chromatography-electrospray ionization mass spectrometry of peptides and proteins using poly(styrene-divinylbenzene) monoliths

Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) utilizing monolithic poly(styrene-divinylbenzene) columns was optimized for the coupling to electrospray ionization mass spectrometry (ESI-MS) by the application of various temperatures and mobile phase additives during peptide and protein analysis. Peak widths at half height improved significantly upon increasing the temperature and ranged from 2.0 to 5.4 s for peptide and protein separations at 70 degrees. Selectivity of peptide elution was significantly modulated by temperature, whereas the effect on proteins was only minor. A comparison of 0.10% formic acid (FA), 0.050% trifluoroacetic acid (TFA), and 0.050% heptafluorobutyric acid (HFBA) as mobile phase additives revealed that highest chromatographic efficiency but poorest mass spectrometric detectabilities were achieved with HFBA. Clusters of HFBA, water, and acetonitrile were observed in the mass spectra at m/z values >500. Although the signal-to-noise ratios for the individual peptides diverged considerably both in the selected ion chromatograms and extracted mass spectra, the average mass spectrometric detectabilities varied only by a factor of less than 1.7 measured with the different additives. Limits of detection for peptides with 500 nl sample volumes injected onto a 60 mm x 0.20 mm monolithic column were in the 0.2-13 fmol range. In the analysis of hydrophobic membrane proteins, HFBA enabled highest separation selectivity at the cost of lower mass spectral quality. The use of 0.050% TFA as mobile phase additive turned out to be the best compromise between chromatographic and mass spectrometric performance in the analysis of peptides and proteins by RP-HPLC-ESI-MS using monolithic separation columns.     

Liquid chromatography with tandem mass spectrometry method for trace level analysis of migrated secondary alkanesulfonate from poly(tetramethylene terephthalate) into food simulants

Secondary alkanesulfonate has been widely used as an antistatic additive in polymers for producing anti‐dust food packaging containers. Currently, no reported method exists for accurate quantification of secondary alkanesulfonate in ethanolic and acidic food simulants. A new liquid chromatography with tandem mass spectrometry method was developed to quantify the migrated amount of secondary alkanesulfonate at trace levels in food simulants from a poly(tetramethylene terephthalate) containing secondary alkanesulfonate. The poly(tetramethylene terephthalate) samples loaded with the antistatic additive were exposed to various food simulants. The collected extracts were directly analyzed by liquid chromatography with tandem mass spectrometry in electrospray ionization negative mode. As secondary alkanesulfonate is a mixture containing C14 to C17 chain lengths, it was separated on a Poroshell 120 EC‐C8 column adopting methanol water gradient program. The migration of secondary alkanesulfonate ranged from 58 to 329 ppb; which is well within the allowed permissible regulatory limits and the adopted method was validated by conducting spiking studies and acceptable recoveries were obtained. The developed method was not only sensitive to detect lower levels of the migrated antistatic additive, but it also avoided more cumbersome sample preparation methodologies like sample enrichment and other derivatization approaches.     

Separation of planar chiral ferrocene derivatives on beta-cyclodextrin-based polymer supports prepared via ring-opening metathesis graft-polymerization

A plastic microfluidic system, containing porous poly(vinylidene fluoride) (PVDF) membranes adsorbed with bovine serum albumin (BSA), is demonstrated for high resolution chiral separation of racemic tryptophan and thiopental mixtures. Microfluidic networks on poly(dimethylsiloxane) (PDMS) substrates are fabricated by capillary molding technique. This miniaturized chiral separation system consists of two layers of PVDF membranes which are sandwiched between two PDMS slabs containing microchannels facing the membranes. On-line adsorption of BSA onto the membranes is employed for the preparation of chiral stationary phase and the evaluation of solution conditions in an effort to achieve maximum protein adsorption. Variations in the mobile phase conditions, including solution pH and ammonium sulfate concentration, are studied for their effects on chiral separation. Based on the large surface area to volume ratio of porous membrane media, adsorbed BSA onto the PVDF membranes enables high resolution separation of racemic mixtures with sample consumption of sub-nanogram or less in the integrated microfluidic networks. In addition, the membrane pore diameter in the submicron range eliminates the constraints of diffusional mass-transfer resistance during protein adsorption and chiral chromatographic processes.     

Coupling on-chip solid-phase extraction to electrospray mass spectrometry through an integrated electrospray tip

We report the integration of solid-phase extraction (SPE) with mass spectrometry (MS) through an on-chip electrospray tip for sample precleaning and preconcentration. An in situ polymerized alkylacrylate-based monolithic column was used as the stationary phase for the on-chip SPE. Each microchip consists of two sets of microchannels and their respective integrated electrospray tips, with a common gold electrode. After the microchip was fabricated from cycloolefin polymer by hot embossing, thermal bonding, and annealing steps, a mixture of monomers and porogenic solvents was pumped into the microchannels and certain areas of the main microchannels were exposed to UV irradiation through a mask. The resulting porous monolithic beds that were polymerized from different compositions of the mixture were characterized by scanning electron microscopy. The microchip containing the monolithic column was then interfaced to an ion trap (IT) mass spectrometer by modifying a commercially available interfacing system. Makeup solution from the side channel was infused concurrently with the solution flowing into the main channel, and the mixture of these two solutions was sprayed into the MS orifice. Both the adsorption and elution of a pharmaceutical test compound, imipramine, to and from the on-chip SPE columns were monitored by MS. The potential application of this device for sample cleanup was demonstrated by pretreatment of urine samples spiked with imipramine.     

Continuous sample deposition from reversed-phase liquid chromatography to tracks on a matrix-assisted laser desorption/ionization precoated target for the analysis of protein digests

Peptide mass fingerprinting by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) is one of the standard high-throughput methods for protein identification today. Traditionally this method has been based on spotting peptide mixtures onto MALDI targets. While this method works well for more abundant proteins, low-abundance proteins mixed with high-abundance proteins tend to go undetected due to ion suppression effects, instrumental dynamic range limitations and chemical noise interference. We present an alternative approach where liquid chromatography (LC) effluent is continuously collected as linear tracks on a MALDI target. In this manner the chromatographic separation is spatially preserved on the target, which enables generation of off-line LC-MS and LC-MS/MS data by MALDI. LC-MALDI sample collection provides improved sensitivity and dynamic range, spatial resolution of peptides along the sample track, and permits peptide mass mapping of low-abundance proteins in mixtures containing high-abundance proteins. In this work, standard and ribosomal protein digests are resolved and captured using LC-MALDI sample collection and analyzed by MALDI-TOF-MS.     

Nickel species analysis of human colonic tissue using liquid chromatography,gel electrophoresis and mass spectrometry

Studies to specify metal-binding species, such as metalloproteins that are present in trace amounts in colonic cell cytosol, using chromatographic separation methods in combination with inductively coupled plasma mass spectrometry (ICP-MS) as element-specific detection require an optimised sample preparation regarding the solubilisation of the proteins. Focus should be taken to avoid metal contamination, enzymatic digestion by different proteases and oxidation. In this article different sample preparation methods are studied to find a suitable method for the isolation and characterisation of Ni species previously found in cytosols from normal and malignant tissues of the human colon. The total Ni concentrations of the cytosols were determined as well as the total protein content. Thus, a Ni-containing protein could be isolated from cytosols of malignant human colonic tissues using size-exclusion chromatography with ICP-MS for element-specific detection. Ni-containing species in the molecular mass range from 10,000 to 20,000 Da were found and pre-concentrated. The determination of the molecular mass of the species was performed through online coupling of reversed-phase chromatography with electrospray ionisation quadrupole time-of-flight MS. Using identical chromatographic conditions and ICP-MS the detected protein was shown to contain Ni.     

Characterization of an opioid peptide-containing protein and of bovine α-lactalbumin by electrospray ionization and liquid secondary ion mass spectrometry

Mass spectrometry methods have been used to characterize two proteins: an opioid peptide-containing protein extracted from bovine pituitary, and bovine α-lactalbumin (BAL). A protein that contains β-endorphin was found in bovine pituitary, and that protein was characterized with electrospray ionization mass spectrometry (ESIMS), gel permeation chromatography, reversed-phase high performance liquid chromatography (RP-HPLC), radioimmunoassay, trypsinolysis, and liquid secondary ion mass spectrometry (LSIMS). BAL is a protein that was used as a model to develop analytical methods to study opioid peptide-containing proteins. Commercial BAL was purified by RP-HPLC, and its molecular weight (M.W.) was determined by ESIMS. The shift in mass observed following dithiothreitol (DTT) reduction estimated the number of disulfide bonds. For all of the data obtained for BAL with or without RP-HPLC separation, ESIMS determined the M.W. of the peptides produced by trypsin treatment of BAL, and LSIMS selected a precursor ion, the protonated molecule ion [M + H]⁺, of a tryptic peptide, which was analyzed by tandem mass spectrometry. Following DTT reduction, ESIMS and LSIMS detected each peptide that contained disulfide bonds in that mixture of tryptic peptides.     

In-line system containing porous polymer monoliths for protein digestion with immobilized pepsin, peptide preconcentration and nano-liquid chromatography separation coupled to electrospray ionization mass spectroscopy

The use of two different monoliths located in capillaries for on-line protein digestion, preconcentration of peptides and their separation has been demonstrated. The first monolith was used as support for covalent immobilization of pepsin. This monolith with well-defined porous properties was prepared by in situ copolymerization of 2-vinyl-4,4-dimethylazlactone and ethylene dimethacrylate. The second, poly(lauryl methacrylate-co-ethylene dimethacrylate) monolith with a different porous structure served for the preconcentration of peptides from the digest and their separation in reversed-phase liquid chromatography mode. The top of the separation capillary was used as a preconcentrator, thus enabling the digestion of very dilute solutions of proteins in the bioreactor and increasing the sensitivity of the mass spectrometric detection of the peptides using a time-of-flight mass spectrometer with electrospray ionization. Myoglobin, albumin, and hemoglobin were digested to demonstrate feasibility of the concept of using the two monoliths in-line. Successive protein injections confirmed both the repeatability of the results and the ability to reuse the bioreactor for at least 20 digestions.     

Capillary columns in liquid chromatography: between conventional columns and microchips

Liquid chromatography on columns with small internal diameters has been reviewed as the intermediate technique between conventional liquid chromatography and microchip separations. The development of micro column separations in the early years has been described, starting with the papers of Horváth and co-workers and Ishii and co-workers, continuing into the first part of the eighties, then making a leap in time to recent innovations with small-bore columns. Based on internal diameters a classification of the different analytical HPLC columns has been suggested. The advantages of small-bore columns have been discussed, with particular emphasis on the advantage of coupling to concentration sensitive detectors when the sample amount is limited. Open tubular columns are treated as a part of the historic background. The recent developments include a brief look into the current status of monolithic columns, the use of packed nano columns and micro columns with electrospray mass spectrometry, and the potential of two-dimensional comprehensive liquid chromatography. Finally, the coupling of sample preparation to analytical columns and the future applications of the novel technological improvements to the microchip separation methods have been discussed.