Particulate capillary precolumns with double-end polymer monolithic frits for on-line peptide trapping and preconcentration

In this work,a novel kind of particulate capillary precolumns with double-end polymer monolithic frits has been developed.Firstly,the polymer monolithic frit at one end was prepared via photo-initiated polymerization of a mixture of lauryl methacrylate and ethyleneglycol dimethacrylate with 1-propanol and 1,4-butanediol as porogens and 2,2-dimethoxy-2-phenylacetophenone as a photo-initiator in UV transparent coating capillary(100 μm i.d.).Subsequently,C18 particles(5 μm,100 A) were packed into the capillary,and sealed with the polymer monolithic frit at another end.To prevent the reaction of monomers and C18 particles,the packed C18 particles were masked during UV exposure.The loading capacity of such a precolumn was determined to be about 9 μg by frontal analysis with a synthetic peptide APGDR1 YVHPF as a model sample.Furthermore,two parallel precolumns were incorporated into a two-dimensional nano-liquid chromatography(2D nano-LC) system with dual capillary trap columns for peptide trapping and concentration.Compared to 2D nano-LC system with a single trap column,such two dimensional separations could be operated simultaneously to improve the analysis throughput.All these results demonstrated that such capillary precolumns with double frits would be promising for high-throughput proteome analysis.     

Integration of immobilized trypsin bead beds for protein digestion within a microfluidic chip incorporating capillary electrophoresis separations and an electrospray mass spectrometry interface

A microfluidic device is described in which an electrospray interface to a mass spectrometer is integrated with a capillary electrophoresis channel, an injector and a protein digestion bed on a monolithic substrate. A large channel, 800 microm wide, 150 microm deep and 15 mm long, was created to act as a reactor bed for trypsin immobilized on 40-60 microm diameter beads. Separation was performed in channels etched 10 microm deep, 30 microm wide and about 45 mm long, feeding into a capillary, attached to the chip with a low dead volume coupling, that was 30 mm in length, with a 50 microm i.d. and 180 microm o.d. Sample was pumped through the reactor bed at flow rates between 0.5 and 60 microL/min. The application of this device for rapid digestion, separation and identification of proteins is demonstrated for melittin, cytochrome c and bovine serum albumin (BSA). The rate and efficiency of digestion was related to the flow rate of the substrate solution through the reactor bed. A flow rate of 1 or 0.5 microL/min was found adequate for complete consumption of cytochrome c or BSA, corresponding to a digestion time of 3-6 min at room temperature. Coverage of the amino acid sequence ranged from 92% for cytochrome c to 71% for BSA, with some missed cleavages observed. Melittin was consumed within 5 s. In contrast, a similar extent of digestion of melittin in a cuvet took 10-15 min. The kinetic limitations associated with the rapid digestion of low picomole levels of substrate were minimized using an integrated digestion bed with hydrodynamic flow to provide an increased ratio of trypsin to sample. This chip design thus provides a convenient platform for automated sample processing in proteomics applications.     

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.     

Voltage-assisted capillary LC of peptides using monolithic capillary columns prepared by ring-opening metathesis polymerization

We examined the use of monolithic capillary columns prepared via ring-opening metathesis polymerization (ROMP) for peptide separation in voltage-assisted capillary LC (voltage-assisted CLC). In order to demonstrate their potential for peptide separation, ROMP-derived monoliths with RP properties were prepared. The preparation procedure of monoliths was transferred from ROMP monoliths optimized for CLC. ROMP monoliths were synthesized within the confines of 200 microm id fused-silica capillaries with a length of 37 cm. After optimization of the chromatographic conditions, the separation performance was tested using a well-defined set of artificial peptides as well as two peptidic mixtures resulting from a tryptic digest of BSA as well as a collagenase digest of collagen. ROMP monoliths showed comparable performance to other monolithic separation media in voltage-assisted CLC published so far. Therefore, we conclude that by optimizing the composition of the ROMP monoliths as well as by using the controlled manner of their functionalization, ROMP monoliths bear a great potential in CLC and CEC.     

Photopolymerized monolithic capillary columns for rapid micro high-performance liquid chromatographic separation of proteins

The preparation of monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) capillary columns using photoinitiated in situ polymerization within 200 microm i.d. capillaries and their application for microHPLC separations of proteins have been studied. The low resistance to flow characteristic of monolithic columns, enabled the use of very high flow rates of up to 100 microL/min representing a flow velocity of 87 mm/s. Very good separations of a model protein mixture consisting of ribonuclease A, cytochrome c, myoglobin, and ovalbumin was achieved in less than 40 s using a very simple single step gradient of the mobile phase. Interestingly, no effect of the pore size on the separations of proteins was observed for these monolithic columns within the size range of 0.66-2.2 microm. The monolithic microHPLC columns are found very robust and no changes in the long term separation performance and back pressure were observed.     

疏水型色谱饼对人血清蛋白质组学样品的快速分离与制备

建立了疏水型色谱饼(10 mm×20 mm i.d.)与反相色谱(RPLC)离线二维色谱快速分离制备人血清蛋白质组学样品,并用基体辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行检测的方法。以4种标准蛋白质的稀溶液为模型进行分离富集,得到细胞色素c(Cyt-c)与肌红蛋白(Myo)的检出限均为1 pmol/μL,溶菌酶(Lys)和胰岛素（Ins）的检出限为0.1 pmol/μL。将此方法用于人血清蛋白质组学样品的分离与制备,随着血清处理量的增大,质谱可检出的组分数目与信号强度均增加,当血清处理量达到1.0 mL时,可检出低丰度蛋白质或多肽285个(相对分子质量均在15000以下)。研究中将1 μg Cyt-c加入到0.5 mL血清中,用上述方法在分离富集低丰度Cyt-c上取得了很好的效果。结果表明,采用疏水型色谱饼与反相色谱联用技术不仅可对血清样品中低丰度蛋白质进行有效的分离和富集,而且一次样品的处理量大,可显著提高低丰度蛋白质的分析、检测水平。     

High performance liquid chromatographic determination of Picumast and two active metabolites in plasma using on-line sample preparation.

A method for determining Picumast, an antiallergic drug, in plasma by HPLC and column switching has been developed. The system consisted of two precolumns, an analytical column, three pumps, an autosampler and a fluorescence detector. The precolumns (17 x 4.6 mm i.d.) were packed with LiChroprep RPR (a moderately polar reversed phase) and the analytical column with Nucleosil ODS (RP 18, 5 microns). The columns were connected according to the alternating precolumn technique. The mobile phase consisted of 30% CH3CN/70% 0.05 M KH2PO4, pH 2.5, with a flow gradient. Detection wavelengths were 333 nm for excitation and 383 nm for emission. The retention times of Picumast, M1 and M2 were 12, 3.6 and 4.0 min, respectively. Total run time was 15 min. The limit of detection was 3 ng/mL for M1 and 1 ng/mL for M2 and Picumast using an injection volume of 150 microL. The recoveries vary between 89% and 97% with standard deviations between 2.4 and 3.3%.     

Application of a high-pressure electro-osmotic pump using nanometer silica in capillary liquid chromatography

A novel designed electro-osmotic pump (EOP) with simple structure was assembled using three 20 cm x 530 microm i.d. fused-silica capillaries packed with 20 +/- 5 nm silica grains for capillary liquid chromatography. It was found that the pump could generate pressures over 20 MPa and several microL/min flow rate for most of the liquids being delivered with the applied voltage less than 10 kV. By increasing the pressure, decreasing the applied voltage and the electrical current, the thermodynamic efficiency was about 1-4%. A practical application of the EOP in a 20cm x 150 microm i.d. 3 microm C18 fused-silica analytical capillary column demonstrated the applicability of the pump.     

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.     

Effects of inner diameter of monolithic column on separation of proteins in capillary-liquid chromatography

Polymer monolithic columns with I.D. between 100 and 320 microm were prepared by in-situ polymerization of styrene and divinylbenzene in fused silica capillaries. The effects of monolithic column I.D. on the separation of proteins in reversed-phase capillary-liquid chromatography under gradient elution were systemically studied. The loading capacity was positively proportional to the volume of the stationary phase. It was found that the smaller diameter columns showed better performance for protein separation. The minimum plate height decreases from 34.99 microm (320 microm I.D. column) to 5.39 microm (100 microm I.D. column) for a retained protein. After studying the three parameters of the Van Deemter equation, it was interpreted that the smaller diameter can provide less flow resistance and the better performance may also be improved by the increasing of the effective diffusion. This conclusion was also supported by the data of separation permeability and breakthrough curves.     

Capillary high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry using a novel nanoflow gradient generator

A type of high-performance liquid chromatography (HPLC) based on a novel nanoflow gradient generator (Asymptotic-Trace-10-Port-Valve (AT10PV) nanoGR generator) was developed and coupled with an electrospray ion trap time-of-flight mass spectrometer (ESI-IT-TOF MS). Stability of the nanoflow GR HPLC system was tested at flow rates of 20 and 50 nL/min by using a nanoflow meter. Average flow rates in a 2-h run were 51.2 nL/min with RSD 0.7% and 21.0 nL/min with RSD 1.8%. Repeatability of analysis of the nanoHPLC/ESI-IT-TOF MS system was also tested by injecting 1.0 microL of trypsin digested bovine serum albumin (BSA) (100 fmol) into a monolithic silica-ODS column (30 microm i.d., 150 mm in length) through a packed silica-ODS trapping column (particle size 5 microm, 150 microm i.d., 10 mm in length). At a flow rate of 50 nL/min, the result demonstrated a reasonably good repeatability of peak retention times (RSD: 0.32-1.1%) and base-ion peak areas (RSD: 4.4-6.6%).     

Nanoliter capillary electrochromatography columns based on collocated monolithic support structures molded in poly(dimethyl siloxane).

Following current trends in miniaturization of analytical chemistry, an inexpensive disposable analytical tool in the form of a liquid chromatography column fabricated on a poly(dimethyl siloxane) (PDMS) chip was created. Ease of fabricating the chromatography column was demonstrated by molding collocated monolithic support structures (COMOSS) directly in the column. Positive photo-resist, SPR 220, was used to create column structures on a negative relief master providing final channel dimensions of 2.7-5.2 microm wide by 10.0 microm deep, while monolithic dimensions were 9.8 x 9.8 x 10.0 microm - 12.3 x 12.3 x 10.0 microm. The ability to separate biological samples such as peptides from a tryptic digest of fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) was shown. Separations in capillary electrochromatographic (CEC) mode were performed yielding column efficiencies of 4.0 x 10(5) plates/m.     

Dual-purpose sample trap for on-line strong cation-exchange chromatography/reversed-phase liquid chromatography/tandem mass spectrometry for shotgun proteomics. Application to the human Jurkat T-cell proteome

A dual-purpose sample-trapping column is introduced for the capacity enhancement of proteome analysis in on-line two-dimensional nanoflow liquid chromatography (strong cation-exchange chromatography followed by reversed-phase liquid chromatography) and tandem mass spectrometry. A home-made dual trap is prepared by sequentially packing C18 reversed-phase (RP) particles and SCX resin in a silica capillary tubing (1.5 cm x 200 microm I.D. for SCX, 0.7 cm x 200 microm for RP) ended with a home-made frit and is connected to a nanoflow column having a pulled tip treated with an end frit. Without having a separate fraction collection and concentration process, digested peptide mixtures were loaded directly in the SCX part of the dual trap, and the SCX separation of peptides was performed with a salt step elution initiated by injecting only 8 microL of NH4HCO3 solution from the autosampler to the dual trap. The fractionated peptides at each salt step were directly transferred to the RP trap packed right next to the SCX part for desalting, and a nanoflow LC-MS-MS run was followed. During the sample loading-SCX fractionation-desalting, flow direction was set to bypass the analytical column to prevent contamination. The entire 2D-LC separation and MS-MS analysis were automated. Evaluation of the technique was made with an injection of 15 microg peptide mixtures from human Jurkat T-cell proteome, and the total seven salt step cycles followed by each RPLC run resulted in an identification of 681 proteins.     

On-line preconcentration of trace carcinogenic polycyclic aromatic hydrocarbons (PAHs) in microcolumn liquid chromatography via large volume injection

The ability and efficiency of micro precolumns made of C30 particles, monolithic silica C18 stationary phase and quartz wool coated with C30, which act as novel solid phase absorbing materials, for the on-line enrichment of aqueous polycyclic aromatic hydrocarbons (PAHs) in microcolumn liquid chromatography (LC) was investigated. The enrichment unit was designed in such a way that micro precolumns were directly connected to a 6-port micro injection valve via fused-silica tubing (0.05 mm I.D.) in order to minimize band broadening of the samples, and the enrichment efficiency of the three materials was tested using 14 PAHs, which are selected by the US Environmental Protection Agency (US EPA), as the analytes. The separation of PAHs was evaluated by using laboratory-made C30 or ODS capillary columns and the results were compared. There were no significant differences showed from the separation of PAHs in terms of peak signal between the C30 and ODS capillary columns, but the C30 capillary column was chosen for the following experiment due to its ability to produce better repeatability than the ODS column. By using the three kinds of precolumn materials, results showed that the precolumn packed with C30 particles as well as the capillary monolithic C18 precolumns (0.1 or 0.2 mm I.D.) provided better recovery than those of the quartz wool's. As long as the recovery and separation of the PAHs were concerned, 0.1 mm I.D. monolithic C18 precolumn showed the best results and the R.S.D.s (N = 7) for the retention time, peak area and peak height were between 0.70-1.5, 2.3-5.8 and 2.4-6.6%, respectively. Large volume injection up to 0.5 mL, i.e. 2500-fold enrichment, was possible and no negative effect on the separation profile was found. The LOD (S/N = 3) were between 0.10 and 4.6 pg mL⁻¹, while the LOQ (S/N = 10) were in the range of 0.32-15 pg mL⁻¹, which showed that the system is comparable to many major analytical techniques and is sensitive enough for the trace analysis of PAHs in environmental samples. The system was then applied to the determination of trace PAHs present in soil sample which was randomly taken from a nearby highway.     

Solvent Trapping during Large Volume Injection with an Early Vapor Exit. Part 3: The Main Cause of Volatile Component Loss during Partially Concurrent Evaporation

When 0.53 mm i.d. uncoated precolumns connected to a solvent vapor exit are used for sample introduction with partially concurrent solvent evaporation, substantial losses of volatile solutes are often observed. They were found to be the consequence of solute accumulation at the front end of the flooded zone, which in turn is the result of a strong pressure drop over the flooded zone owing to the formation of plugs of sample liquid. The pressure drop causes significant solvent evaporation at the front, which enriches the solute material there and causes its loss. The use of 0.32 mm i.d. restrictions between the uncoated precolumn and the vapor exit greatly reduced this problem.     

Stability of Parathion-Methyl,Methiocarb, DDT and 2,4-D in Solid-Phase Extraction Precolumns Packed with a Polymeric Reversed Phase

Abstract

The stability of parathion-methyl, methiocarb, DDT and 2,4-D, adsorbed on the polymeric PLRP-S phase packed in small stainless steel precolumns was examined, with a view to propose the use of these precolumns as alternative means for the transport of water samples. First, water samples spiked with the studied pesticide at low μg/l concentration levels were extracted and preconcentrated in the precolumns, using appropriate conditions for a total recovery. Then, the precolumns were stored at room temperature (15—20°C) or at 35°C for different time periods. At the end of the respective period each precolumn was coupled to an HPLC column via a switching valve and was on-line analyzed by reversed phase chromatography with UV detection. The four pesticide recoveries after one week in the precolumn at room temperature were higher than 90%. The same was true at 35°C except for DDT, which suffered a 30% degradation in one week. Further studies showed that DDT and parathion-methyl were stable at least for five weeks in precolumns stored at room temperature. Methiocarb also was stable for this period but storing the precolumns at 4°C.     

Gas chromatographic retention in uncoated fused silica capillaries

Understanding of retention in uncoated fused‐silica capillaries is of interest due to increased attention on precolumn backflushing in capillary GC. Uncoated capillaries offer several advantages as precolumns compared to coated precolumns. In order to examine the possibility of predicting elution temperatures of alkanes from uncoated capillaries a priori, several sizes of deactivated but uncoated fused‐silica capillaries were evaluated under various operating conditions. Retention was found to depend on dimensionless ramp rate (°C/t_M), sample loading (capacity), flow mode, and column dimensions (probably related to surface area).     

Evaluation of ring-opening metathesis polymerization (ROMP)-derived monolithic capillary high performance liquid chromatography columns

Novel monolithic capillary HPLC columns were prepared via ring opening metathesis polymerization (ROMP) within the confines of fused silica columns with 200 microm i.d. using norborn-2-ene (NBE), 1,4,4a,5,8,8a-hexahydro-1,4,5,8, exo, endo-dimethanonaphthalene (DMN-H6) as monomers, 2-propanol and toluene as porogens and RuCl2(PCy3)2(CHPh) as initiator. Using the monolithic capillary HPLC columns, different sets of analytes (i.e. standard systems) were used for the evaluation of the monolithic columns: (i) a protein standard consisting of six proteins in the range of 5000-66 000 g/mol, (ii) an insulin-albumin standard, and (iii) a peptide standard obtained from a tryptic digest of cytochrome C. With these three different standard systems the reproducibility of synthesis in terms of separation performance proved to be 1-2% relative standard deviation in tR. Variation of polymerization parameters had a significant influence on the monolithic morphology and therefore separation efficiency and back pressure. The maximum analytical loading capacity of ROMP-derived monolithic capillary columns for albumin was found to be 30-125 ng, depending on the monomer content. Long-term stability studies showed no alteration in separation performance.     

Design, optimisation, and evaluation of a sheath flow interface for automated capillary electrophoresis-electrospray-mass spectrometry

A sheath-flow capillary electrophoresis-mass spectrometry (CE-MS) system utilizing a fully integrated large-bore stainless-steel emitter electrode tapered at the end for micro-ionspray operation has been developed and evaluated. A separation capillary with an outer diameter of up to 360 microm was inserted into the electrode thus forming a void volume of less than 15 nL between the capillary end and the electrospray ionisation (ESI) tip. The sheath liquid, usually methanol-water (80:20) with 0.1% formic acid for positive ion mode or methanol for negative ion mode, was delivered at 0.5-1.0 microL/min. Unlike previously reported CE-MS interfaces, the CE-MS probe was incorporated directly onto an Applied Biosystems/MDS SCIEX orthogonal-spray Turbo "V" ion source for ease of use and automatic operation. This integration enables fast and facile coupling and replacement of the separation capillary without interrupting the ion source configuration, and the sheath liquid supply. The reusable electrospray electrode was precisely fabricated and aligned with the length of the nebulizing gas tube for improved reproducibility. Automation was achieved through software control of both CE and tandem MS (MS/MS) for unattended batch sample analysis. The system was evaluated for attomole- to low femtomole-level profiling of model peptides and protein mixtures, bisphosphates, as well as antiviral nucleosidic drugs in cellular extracts.     

Enrichment of peptides from plasma for peptidome analysis using multiwalled carbon nanotubes

Human plasma contains a complex matrix of proteolytically derived peptides (plasma peptidome) that may provide a correlate of biological events occurring in the entire organism. Analyzing these peptides from a small amount of serum/ plasma is difficult due to the complexity of the sample and the low levels of these peptides. Here, we describe a novel peptidome analysis approach using multiwalled carbon nanotubes (MWCNTs) as an alternative adsorbent to capture endogenous peptides from human plasma. Harvested peptides were analyzed by using liquid chromatography-mass spectrometry as a means of detecting and assessing the adsorbed molecules. The improved sensitivity and resolution obtained by using liquid chromatography-mass spectrometry allowed detection of 2521 peptide features (m/z 300-1800 range) in about 50 microL of plasma. 374 unique peptides were identified with high confidence by two-dimensional liquid chromatography system coupled to a nano-spray ionization linear ion trap-mass spectrometer. High recovery of BSA digest peptides enriched with MWCNTs, in both standard buffer and high abundance protein solution, was observed. Comparative studies showed that MWCNTs were superior to C18 and C8 for the capture of the smaller peptides. This approach could hold promise of routine plasma peptidome analysis.