Efficient protein digestion with peptide separation in a micro-device interfaced to electrospray mass spectrometry

A highly efficient protein digestion device has been fabricated using commercially available immobilized trypsin on agarose beads, packed into a silica capillary and connected either directly to an electrospray mass spectrometer via a 'microtight T' connector, from which aqueous acetic acid (0.2%) was pumped, or via a monolithic column connected to the mass spectrometer ion source. Six proteins with molecular mass ranging from 2848 to 77703 Da were digested completely using this system. In the second set of experiments a short monolithic separation column was placed after the immobilized trypsin capillary and partial separation of the generated peptides was obtained. The detection limits were increased from the micromol to pmol range by utilization of this separation column. Gradient elution, using a binary HPLC pump and a flow splitter, was used to optimize the peptide separation. This provided significantly enhanced resolution of the tryptic peptides but increased the analysis time to 30 minutes.     

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

Recent progress in adsorbed stationary phases for capillary electrochromatography

This review surveys the recent progress in the adsorbed stationary phases for capillary electrochromatography (CEC). Adsorption-based methods for preparation of stationary phase are novel approaches in CEC, which allow rapid and facile preparing stationary phases with desirable selectivity onto an open-tubular fused-silica capillary, a bare-silica or ion-exchange packed column or a monolithic silica or polymer column. A variety of adsorbing agents have been developed as adsorbed stationary phases, including ionic long-chain surfactant, protein, peptide, amino acid, charged cyclodextrin (CD), basic compound, aliphatic ionene, and ion-exchange latex particle. The adsorbed stationary phases have been applied to separation of neutral, basic and acidic organic compounds, inorganic anions and enantiomers. They have also been applied to on-line sample concentration, fast separation and study of the competitive binding of enantiomers with protein.     

Monolithic silica-based capillary column with strong chiral cation-exchange type surface modification for enantioselective non-aqueous capillary electrochromatography

A silica-based monolithic stationary phase prepared by the sol-gel process in a 100 microm I.D. fused-silica (FS) capillary has been modified chemically with 3-mercaptopropyl trimethoxysilane followed by immobilization of a strong cation-exchange (SCX) type chiral selector, (S)-N-(4-allyloxy-3,5-dichlorobenzoyl)-2-amino-3,3-dimethylbutane phosphonic acid, by radical addition reaction onto the reactive sulfhydryl surface. After a fine-tuning of the mobile phase composition, the enantioselective capillary column was evaluated for the separation of various chiral basic drugs by enantioselective non-aqueous capillary electrochromatography (CEC), in comparison to capillary column analogs packed with 3.5 microm silica particles having attached the same selector. The performance of the monolithic silica column was further compared to corresponding polymethacrylate-based organic polymer monoliths. The study indicated that strong counter-ions such as 2-aminobutanol or N,N,N',N'-tetramethylethylenediamine are needed, although they reduce the electroosmotic flow velocity and separation factors in comparison to less efficient counter-ions, in order to allow the elution of the oppositely charged solutes in the ion-exchange retention mode within reasonable run time and as sharp zones. In contrast, weak counter-ions such as N,N-diisopropylethylamine (Huenig base) provided stronger electroosmotic flow and much better separation factors, but relatively poor peak efficiencies. Overall, with the chemically functionalized monolithic silica column the high quality separations of packed column analogs could be approximated, with regards to both separation factors and peak performances. On the other hand, the monolithic capillary column certainly outperformed the packed column in terms of system robustness under capillary electrochromatography conditions and showed excellent column longevity. The enantioselective strong cation-exchange-type monolithic silica column performed also well in comparison to the organic polymer monolith.     

Capillary high-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic columns and carbon fiber electrospray ionization emitters

Monolithic columns having long hydrocarbon chains were prepared by in-situ polymerization in capillary fused silica tubing. The capillary columns were coupled with a newly developed carbon fiber electrospray ionization (ESI) emitter for proteomic analysis using sheathless capillary HPLC-ESI mass spectrometry (MS). The sample loading capacity and chromatographic performance of the styrene-based monolithic column, which was prepared by photo-polymerization of octylstyrene (OS) and divinylbenzene (DVB) were compared with that of the methacrylate-based monolithic column composed of lauryl methacrylate (LMA) and ethylene dimethacrylate (EDMA). The sample loading ability of tryptic digested protein in poly-OS (POS)-DVB column was higher than that of poly-LMA (PLMA)-EDMA column, possibly due to the irregular and rugluous surface offering a greater surface area of POS-DVB stationary phase. The POS-DVB column also provided better separation efficiency in the separation of high concentration (10 microg) of tryptic digested albumin bovine serum (BSA). Due to the successful interface of a highly efficient monolithic column and a stable, durable carbon fiber emitter, low femtomole levels of peptides were successfully separated and identified in the presence of large amounts of tryptic digested protein.     

Characterization of some physical and chromatographic properties of monolithic poly(styrene-co-divinylbenzene) columns

Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200 microm i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. Important chromatographic features of the synthesized columns were characterized and critically compared to the properties of columns packed with micropellicular, octadecylated poly(styrene-co-divinylbenzene) (PS-DVB-C18) particles. The permeability of a 60 mm long monolithic column was slightly higher than that of an equally dimensioned column packed with PS-DVB-C18 beads and was invariant up to at least 250 bar column inlet pressure, indicating the high-pressure stability of the monolithic columns. Interestingly, monolithic columns showed a 3.6 times better separation efficiency for oligonucleotides than granular columns. To study differences of the molecular diffusion processes between granular and monolithic columns, Van Deemter plots were measured. Due to the favorable pore structure of monolithic columns all kind of diffusional band broadening was reduced two to five times. Using inverse size-exclusion chromatography a total porosity of 70% was determined, which consisted of internodule porosity (20%) and internal porosity (50%). The observed fast mass transfer and the resulting high separation efficiency suggested that the surface of the monolithic stationary phase is rather rough and does not feature real pores accessible to macromolecular analytes such as polypeptides or oligonucleotides. The maximum analytical loading capacity of monolithic columns for oligonucleotides was found to be in the region of 500 fmol, which compared well to the loading capacity of the granular columns. Batch-to-batch reproducibility proved to be better with granular stationary phases compared to monolithic stationary phase, in which each column bed is the result of a unique column preparation process.     

Recent advances in monolithic columns for protein and peptide separation by capillary liquid chromatography

Capillary liquid chromatography (cLC) has great potential for protein and peptide separation, with advantages of high efficiency, high resolution, low sample consumption, and high sensitivity when coupled with mass spectrometry. In recent years, monoliths have been widely used as the stationary phases for capillary columns, owing to easy preparation, high permeability, fast mass transfer, and low backpressure. This review summarizes recent advances (2007–2012) in monolithic columns for protein and peptide separation by cLC. After a brief introduction on the preparation of monolithic capillary columns, the emphasis of this review is focused on the recent application of such columns for protein and peptide separation by cLC. Furthermore, the challenges and potential hot points of monolithic capillary columns in the future are discussed.     

Rapid separation and sensitive detection method for beta-blockers by pressure-assisted capillary electrochromatography-electrospray ionization mass spectrometry

A new method for rapid separation and sensitive detection of beta-blockers by pressure-assisted capillary electrochromatography (pCEC) with electrospray ionization mass spectrometry (ESI-MS) using silica-based monolithic column was studied in this paper. The proposed method has been confirmed to be very powerful since the fast mass transfer property and good permeability of silica monolithic column was used in this pCEC-ESI-MS system. In this work, a silica monolithic column was prepared with sol-gel method for simultaneous fast separation of beta-blockers. Furthermore, in order to obtain the highly selective and sensitive result of pCEC-ESI-MS, both the CEC separation and MS detection parameters were optimized in detail. Under the optimized conditions, namely 80% acetonitrile and 20% 20 mmol/L ammonium acetate (pH 6.0) as the mobile phase, 20 kV and 8 bar as the separation voltage and the assisted pressure, isopropanol/water (1:1, v/v) containing 7.5 mmol/L acetic acid as the sheath liquid, and 3 microL/min as the flow rate of sheath liquid, seven beta-blockers were well separated within 11 min with detection limits in the range of 0.15-0.80 ng/mL (defined as S/N=3). The recoveries of spiked urine samples of these beta-blockers were between 86.3 and 103% with the RSDs lower than 8.0%. The real samples from some male volunteers were successfully analyzed and confirmed with the proposed method. Comparing with GC-MS or LC-MS, the new method has some superiority (such as fast analysis capacity and simple pretreatment) in clinical practice and doping control.     

Hydrophilic interaction chromatography coupled to electrospray mass spectrometry for the separation of peptides and protein digests

In this study, the analysis of a peptide set, chosen for their differences in hydrophilicity, and the tryptic digests of bovine cytochrome c and β-lactoglobulin by hydrophilic interaction chromatography–electrospray ionisation mass spectrometry (HILIC–ESI-MS) is demonstrated. Two different types of HILIC phases, i.e., an amide- and an amino-modified silica-based phase, packed into narrow bore or capillary columns, were investigated with separations conducted under either low pH or neutral pH conditions. The separation performance of the two HILIC columns with respect to peak efficiency and selectivity have been documented under these different mobile phase conditions, and the results compared with the performance of a conventional capillary reversed-phase C18 column of similar dimensions. It was found that very good separation of the peptide set could be achieved by using the amide-modified silica column over a broad pH range. Moreover, with the protein digest samples, excellent separation of the tryptic digests was obtained with the amide-modified HILIC column under neutral pH conditions. Compared to the conventional reversed-phase C18 separations, the use of these HILIC columns not only provided complementary separation selectivity, but also offered the capability to identify unique peptides using tandem HILIC–mass spectrometric techniques. These studies therefore highlight the potential of HILIC procedures for future proteomic applications.     

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.     

Selectivity tuning via temperature pulsing using low thermal mass liquid chromatography and monolithic columns

Low thermal mass LCwas applied to the capillary LC separation of a complex insecticide mixture by increasing temperature and decreasing gradients, as well as fast selected temperature pulses to increase resolution of overlapped components. The technology was applied using a new generation of capillary monolithic stationary phases. Considerable peak shifts and selectivity changes were observed for given temperature conditions. The concept of temperature pulsing during an elution profile shows promise for increasing resolution in difficult separations and can provide a relatively simple means to solve coelution problems.     

Detection of low-abundance impurities in synthetic thyroid hormones by stationary phase optimized liquid chromatography–mass spectrometry

The transfer of a gradient method to an isocratic or multistep gradient method employing stationary phase optimized liquid chromatography facilitated a reduction in analysis time by 50% and significantly improved the mass spectrometric detectability of impurities in synthetic thyroid hormones. Four column segments packed with different stationary phases were combined into a single chromatographic column, which allowed the separation and photometric as well as mass spectrometric detection of thyroid compounds in less than 30 min under isocratic- or step gradient elution conditions with 0.10% acetic acid/acetonitrile. Signal instability and baseline drift during detection by negative electrospray ionization time-of-flight mass spectrometry were minimized by optimizing the spray parameters for each individual elution step. This resulted in improved detectabilities and higher mass spectral quality, especially for low-abundance components in the sample mixture. The method was applied to the separation and detection of the low-abundance impurities formed upon the thermal stressing of a sample of synthetic levothyroxine.     

Polymetacrylate and hybrid interparticle monolithic columns for fast separations of proteins by capillary liquid chromatography

Preparation of organic polymer monolithic columns in fused silica capillaries was aimed at fast gradient separation of proteins. For this purpose, polymerization in situ procedure was optimized, using ethylene dimetacrylate and butyl metacrylate monomers with azobisisobutyronitrile as initiator of the polymerization reaction in presence of non-aqueous porogen solvent mixtures composed of 1-propanol and 1,4-butanediol. The separation of proteins in totally monolithic capillary columns was compared with the chromatography on a new type of "hybrid interparticle monolithic" capillary columns, prepared by in situ polymerization in capillary packed with superficially porous spherical beds, 37-50 microm. The "hybrid" columns showed excellent stability and improved hydrodynamic flow properties with respect to the "totally" monolithic capillary columns. The separation selectivity is similar in the two types of columns. The nature of the superficially porous layer (bare silica or bonded C18 ligands) affects the separation selectivity less significantly than the porosity (density) of the monolithic moiety in the interparticle space, controlled by the composition of the polymerization mixture. The retention behaviour of proteins on all prepared columns is consistent with the reversed-phase gradient elution theory.     

Preparation of a butyl–silica hybrid monolithic column with a “one-pot” process for bioseparation by capillary liquid chromatography

A butyl–silica hybrid monolithic column for bioseparation by capillary liquid chromatography (cLC) was prepared with butyl methacrylate and alkoxysilanes through a “one-pot” process. The effects of polycondensation temperature, volume percentage of N,N′-dimethylformamide, and content of cetyltrimethylammonium bromide and butyl methacrylate on the morphologies of the hybrid monolithic columns prepared were investigated in detail. Baseline separations of proteins and small peptides on the hybrid monolithic column were achieved by cLC with gradient elution. In addition, the resulting hybrid column was also applied for analysis of tryptic digests of bovine serum albumin by cLC coupled with tandem mass spectrometry. The results demonstrate its potential application in separation of complex biological samples.     

Open-tubular capillary electrochromatography-mass spectrometry with sheathless nanoflow electrospray ionization for analysis of amino acids and peptides

A novel, rugged sheathless capillary electrochromatography-electrospray ionization (CEC-ESI) device, in which an open-tubular separation capillary and an electrospray tip are integrated with a Nafion tubing junction, is coupled to mass spectrometry (MS) for the analysis of amino acids and peptides. A stable electrospray was generated at nanoflow rates by applying a positive electrical potential at the Nafion membrane junction. To sustain the stable spray, an electroosmotic flow (EOF) to the spray was supported by coating the fused silica capillary with Lupamin, a high-molecular-weight linear positively charged polyvinylamine (PVAm) polymer, which also minimizes analyte adsorption. Electrochromatographic separation of amino acids and peptides was further enhanced by the chromatographic selectivity of Lupamin stationary phase for these molecules. The device was very reliable and reproducible for CEC-ESI-MS analyses of amino acids and peptides for over a hundred injections. The separation and detection behaviors of amino acids and peptides under different conditions including pH, concentration, and composition of mobile phases on Lupamin-coated and uncoated capillaries have been investigated. The relationship between nano electrospray stability and EOF is discussed.     

酯型十八烷基键合硅胶整体柱的制备、表征及性能评价

将硅烷偶联剂γ-(2,3-环氧丙氧)丙基三甲氧基硅烷与十八酸反应,再键合到硅胶整体柱上,得到了酯型十八烷基键合固定相,并用红外光谱、元素分析对其进行了表征。在以甲醇-水为流动相的反相色谱条件下分离了苯、联苯和蒽的混合样品,评价了该整体柱的色谱性能,考察了该整体柱适用的pH范围,以及柱压降、柱效与流速的关系。结果表明,该硅胶整体柱键合效果良好,具有较好的反相色谱性能,且在pH=2～8时稳定性好,柱压降、柱效受流速影响较小,可有效地用于化合物的快速分离分析。     

Study of triacontyl-functionalized monolithic silica capillary column for reversed-phase capillary liquid chromatography

A novel stationary phase triacontyl-functionalized monolithic silica capillary column was successfully prepared for reversed-phase capillary liquid chromatography. The performance of the monolithic silica capillary column coated with triacontyl chain for the separation of alkylbenzenes, xylene isomers, polycyclic aromatic hydrocarbons, and mixture of α- and β-carotenes was studied, which was compared to that using the monolithic silica capillary column coated with octadecyl chain. The comparison results showed that triacontyl-functionalized monolithic silica capillary column would be a promising media to be used for the separation of isomeric solutes with long chain in reversed-phase capillary liquid chromatography.     

Recent progress in enantiomer separation by capillary electrochromatography

Enantiomer separation by electrochromatography (CEC) can be performed in three modes: (i) open-tubular capillary electrochromatography (o-CEC), in which the chiral selector is physically adsorbed coated, and thermally immobilized or covalently attached to the internal capillary wall; (ii) packed capillary electrochromatography (p-CEC), in which the capillary is either filled with chiral modified silica particles or with an achiral packing material, and a chiral selector is added to the mobile phase; and (iii) monolithic (rod)-capillary electrochromatography (rod-CEC) in which the chiral stationary phase (CSP) consists of a single piece of porous solid. We present an overview on methods and new trends in the field of electrochromatographic enantiomer separation such as CEC with either nonaqueous mobile phases or stationary phases with incorporated permanent charges, or with packing beds consisting of nonporous silica particles or particles with very small internal diameters.     

Short communication performance of octadecylsilylated monolithic silica capillary columns of 530 microm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.     

Recent developments in the field of monolithic stationary phases for capillary electrochromatography

This review summarizes the contributions to the rapidly growing area of monolithic columns based on both silica and synthetic polymers for capillary electrochromatography and chip electrochromatography, with a focus on those published during the year 2004. A wide variety of both modified approaches to the "old" monoliths and new monoliths have been reported despite the very short period of time covered. This demonstrates that monolithic stationary phases have become a well-established format in the field of electrochromatography. The simplicity of their preparation as well as the good control over their porous properties and surface chemistries make the monolithic separation media an attractive alternative to capillary columns packed with particulate materials.