Microfluidic gradient formation for nanoflow chip LC

We report a method for forming a nanoflow liquid chromatography (nano-LC) gradient using a single fluid pump at flow rates below 1 muL/min by passively forming a gradient on a microfluidic device. This device works together with an Agilent HPLC-Chip to perform high-throughput nanoflow liquid chromatography/mass spectrometry (nano-LC/MS). The nanoflow gradient delay time is reduced from several minutes for a commercial LC nanoflow pump to only a few seconds with this microfluidic device, thus shortening the total analysis time and increasing the analysis throughput. With this microfluidic device, a nano-LC solvent delivery system can be greatly simplified and have increased robustness, reliability, reduced waste, and ease of use.     

Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns

Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel automated method for the enrichment of phosphopeptides from complex mixtures. The method employs a two-dimensional column setup, with titanium oxide-based solid-phase material (Titansphere) as the first dimension and reversed-phase material as the second dimension. Phosphopeptides are separated from nonphosphorylated peptides by trapping them under acidic conditions on a TiO(2) precolumn. Nonphosphorylated peptides break through and are trapped on a reversed-phase precolumn after which they are analyzed by nanoflow LC-ESI-MS/MS. Subsequently, phosphopeptides are desorbed from the TiO(2) column under alkaline conditions, reconcentrated onto the reversed-phase precolumn, and analyzed by nanoflow LC-ESI-MS/MS. The selectivity and practicality of using TiO(2) precolumns for trapping phosphopeptides are demonstrated via the analysis of a model peptide RKISASEF, in a 1:1 mixture of a non- and a monophosphorylated form. A sample of 125 fmol of the phosphorylated peptide could easily be isolated from the nonphosphorylated peptide with a recovery above 90%. In addition, proteolytic digests of three different autophosphorylation forms of the 153-kDa homodimeric cGMP-dependent protein kinase are analyzed. From proteolytic digests of the fully autophosphorylated protein at least eight phosphorylation sites are identified, including two previously uncharacterized sites, namely, Ser-26 and Ser-44. Ser-26 is characterized as a minor phosphorylation site in purified PKG samples, while Ser-44 is identified as a novel in vitro autophosphorylation target. These results clearly show that TiO(2) has strong affinity for phosphorylated peptides, and thus, we conclude that this material has a high potential in the field of phosphoproteomics.     

On-line 1D and 2D porous layer open tubular/LC-ESI-MS using 10-microm-i.d. poly(styrene-divinylbenzene) columns for ultrasensitive proteomic analysis

Following on our recent work, on-line one-dimensional (1D) and two-dimensional (2D) porous layer open tubular/liquid chromatography-electrospray ionization-mass spectrometry (PLOT/LC-ESI-MS) platforms using 3.2 mx10 microm i.d. poly(styrene-divinylbenzene) (PS-DVB) PLOT columns have been developed to provide robust, high-performance, and ultrasensitive proteomic analysis. With the use of a PicoClear tee, the dead volume connection between a 50 microm i.d. PS-DVB monolithic micro-SPE column and the PLOT column was minimized. The micro-SPE/PLOT column assembly provided a separation performance similar to that obtained with direct injection onto the PLOT column at a mobile phase flow rate of 20 nL/min. The trace analysis potential of the platform was evaluated using an in-gel tryptic digest sample of a gel fraction (15-40 kDa) of a cervical cancer (SiHa) cell line. As an example of the sensitivity of the system, approximately 2.5 ng of protein in 2 microL of solution, an amount corresponding to 20 SiHa cells, was subjected to on-line micro-SPE-PLOT/LC-ESI-MS/MS analysis using a linear ion trap MS. A total of 237 peptides associated with 163 unique proteins were identified from a single analysis when using stringent criteria associated with a false positive rate of less than 1%. The number of identified peptides and proteins increased to 638 and 343, respectively, as the injection amount was raised to approximately 45 ng of protein, an amount corresponding to 350 SiHa cells. In comparison, only 338 peptides and 231 unique proteins were identified (false positive rate again less than 1%) from 750 ng of protein from the identical gel fraction, an amount corresponding to 6000 SiHa cells, using a typical 15 cmx75 microm i.d. packed capillary column. The greater sensitivity, higher recovery, and higher resolving power of the PLOT column resulted in the increased number of identifications from only approximately 5% of the injected sample amount. The resolving power of the micro-SPE/PLOT assembly was further extended by 2D chromatography via combination of the high-efficiency reversed-phase PLOT column with strong cation-exchange chromatography (SCX). As an example, 1071 peptides associated with 536 unique proteins were identified from 75 ng of protein from the same gel fraction, an amount corresponding to 600 cells, using five ion-exchange fractions in on-line 2D SCX-PLOT/LC-MS. The 2D system, implemented in an automated format, led to simple and robust operation for proteomic analysis. These promising results demonstrate the potential of the PLOT column for ultratrace analysis.     

Nanoflow gradient generator coupled with mu-LC-ESI-MS/MS for protein identification

The large-scale identification of proteins from proteomes of complex organisms, and the availability of various types of protein and DNA databases, increasingly require the additional information provided by tandem mass spectrometry. HPLC and microLC coupled to ESI-MS/MS presently dominate the field of protein identification by tandem mass spectrometry and database searching. The analysis of protein digests is typically performed using HPLC or LC columns with 50-100-microm diameters, requiring the delivery of solvent gradients at low to mid nanoliter per minute flow rates. This has been typically achieved using expensive generic HPLC pumping systems for the delivery of microliter per minute gradients that were either flow-split or sampled. Here we present an alternative system for the delivery of nanoliter per minute gradients. The inexpensive nanoflow gradient generator (etagrad) described here can be modulated to reproducibly deliver selected gradients. The performance of the etagrad on-line with a microLC-ESI-MS/MS system has been demonstrated for the identification of standard protein digests. Moreover, the performance of the etagrad-microLC-ESI-MS/MS system, with protein prefractionation by IPG isoelectric focusing, was also evaluated for rapid study of yeast and human proteomes.     

High-efficiency on-line solid-phase extraction coupling to 15-150-microm-i.d. column liquid chromatography for proteomic analysis

The ability to manipulate and effectively utilize small proteomic samples is important for analyses using liquid chromatography (LC) in combination with mass spectrometry (MS) and becomes more challenging for very low flow rates due to extra column volume effects on separation quality. Here we report on the use of commercial switching valves (150-microm channels) for implementing the on-line coupling of capillary LC columns operated at 10,000 psi with relatively large solid-phase extraction (SPE) columns. With the use of optimized column connections, switching modes, and SPE column dimensions, high-efficiency on-line SPE-capillary and nanoscale LC separations were obtained demonstrating peak capacities of approximately 1000 for capillaries having inner diameters between 15 and 150 microm. The on-line coupled SPE columns increased the sample processing capacity by approximately 400-fold for sample solution volume and approximately 10-fold for sample mass. The proteomic applications of this on-line SPE-capillary LC system were evaluated for analysis of both soluble and membrane protein tryptic digests. Using an ion trap tandem MS it was typically feasible to identify 1100-1500 unique peptides in a 5-h analysis. Peptides extracted from the SPE column and then eluted from the LC column covered a hydrophilicity/hydrophobicity range that included an estimated approximately 98% of all tryptic peptides. The SPE-capillary LC implementation also facilitates automation and enables use of both disposable SPE columns and electrospray emitters, providing a robust basis for automated proteomic analyses.     

Microfluidic platform for liquid chromatography-tandem mass spectrometry analyses of complex peptide mixtures

A microfluidic chip that integrates all the fluidic components of a gradient liquid chromatography (LC) system is described. These chips were batch-fabricated on a silicon wafer using photolithographic processes and with Parylene as the main structural material. The fabricated chip includes three electrolysis-based electrochemical pumps, one for loading the sample and the other two for delivering the solvent gradient; platinum electrodes for delivering current to the pumps and establishing the electrospray potential; a low-volume static mixer; a column packed with silica-based reversed-phase support; integrated frits for bead capture; and an electrospray nozzle. The fabricated structures were able to withstand pressures in excess of 250 psi. The device was used to perform a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a mixture of peptides from the trypsin digestion of bovine serum albumen (BSA). Gradient elution through the 1.2-cm column was performed at a flow rate of 80 nL/min. Compared to the analysis of the same sample using a commercial nanoflow LC system, the chromatographic resolution was nearly as good, and the total cycle time was significantly reduced because of the minimal volume between the pumps and the column. Results demonstrate the potential of mass-produced, low-cost microfluidic systems capable of performing LC separations for proteomics applications.     

Microscale phosphoproteome analysis of 10,000 cells from human cancer cell lines

We developed a miniaturized LC-MS system with a high-recovery phosphopeptide enrichment protocol that allows phosphoproteome analysis of 10(4) cells. In the enrichment protocol, the key step is to add sodium deoxycholate and sodium lauroyl sarcosinate to the buffer solution for protein extraction and digestion and to omit any subsequent desalt/desurfactant step before phosphopeptide enrichment. The phosphopeptides enriched by hydroxy acid-modified metal oxide chromatography (HAMMOC) are directly injected onto a miniaturized LC column using a nitrogen-pressure-driven cell, instead of switching valve-type injectors. The miniaturized analytical column of 25 μm diameter provided a 3.6-fold improvement in sensitivity over the conventional 100 μm diameter column. Overall, our analytical system provided approximately 80-fold improvement on average in the LC-MS response, and we identified 1011 unique phosphorylated sites based on 995 unique phosphopeptides from a single analysis of 10(4) HeLa cells (approximately 1 μg of proteins). This is the most sensitive phosphoproteomics system that has so far been reported for proteome-wide analysis of in vivo phosphorylation in mammalian cells.     

Online nanoflow reversed phase-strong anion exchange-reversed phase liquid chromatography-tandem mass spectrometry platform for efficient and in-depth proteome sequence analysis of complex organisms

The dynamic range of protein expression in complex organisms coupled with the stochastic nature of discovery-driven tandem mass spectrometry (MS/MS) analysis continues to impede comprehensive sequence analysis and often provides only limited information for low-abundance proteins. High-performance fractionation of proteins or peptides prior to mass spectrometry analysis can mitigate these effects, though achieving an optimal combination of automation, reproducibility, separation peak capacity, and sample yield remains a significant challenge. Here we demonstrate an automated nanoflow 3-D liquid chromatography (LC)-MS/MS platform based on high-pH reversed phase (RP), strong anion exchange (SAX), and low-pH reversed phase (RP) separation stages for analysis of complex proteomes. We observed that RP-SAX-RP outperformed RP-RP for analysis of tryptic peptides derived from Escherichia coli and enabled identification of proteins present at a level of 50 copies per cell in Saccharomyces cerevisiae, corresponding to an estimated detection limit of 500 amol, from 40 μg of total lysate on a low-resolution 3-D ion trap mass spectrometer. A similar study performed on a LTQ-Orbitrap yielded over 4000 unique proteins from 5 μg of total yeast lysate analyzed in a single, 101 fraction RP-SAX-RP LC-MS/MS acquisition, providing an estimated detection limit of 65 amol for proteins expressed at 50 copies per cell.     

Nanoflow gradient generator for capillary high-performance liquid chromatography

A novel method of generating a nanoflow gradient elution for a capillary high-performance liquid chromatography (HPLC) system has been developed. An important feature of this system is that any gradient (GR) profile generated by a conventional microflow GR pump can be asymptotically traced and converted as a corresponding nanoflow GR profile simply by using a 10-port switching valve with two injection loops installed. Consequently, it has been called an "asymptotic trace 10-port valve" (AT10PV) nanoflow GR generator. Performance of the AT10PV nanoflow GR generator was tested in the range of flow rates from 50 to 500 nL/min. The test demonstrated that the AT10PV nanoflow GR generator can asymptotically trace the original gradient profile with good reproducibility. A capillary HPLC system using the AT10PV nanoflow GR generator provides reasonably good repeatability of peak retention times on the chromatogram of the tryptic digest of a BSA sample, RSD of less than 0.3% at a flow rate of 200 nL/min. It also enables sequential running of a series of sample injections in the same manner as conventional analysis at microflow rates.     

Top-down identification and quantification of stable isotope labeled proteins from Aspergillus flavus using online nano-flow reversed-phase liquid chromatography coupled to a LTQ-FTICR mass spectrometer

Online liquid chromatography-mass spectrometric (LC-MS) analysis of intact proteins (i.e., top-down proteomics) is a growing area of research in the mass spectrometry community. A major advantage of top-down MS characterization of proteins is that the information of the intact protein is retained over the vastly more common bottom-up approach that uses protease-generated peptides to search genomic databases for protein identification. Concurrent to the emergence of top-down MS characterization of proteins has been the development and implementation of the stable isotope labeling of amino acids in cell culture (SILAC) method for relative quantification of proteins by LC-MS. Herein we describe the qualitative and quantitative top-down characterization of proteins derived from SILAC-labeled Aspergillus flavus using nanoflow reversed-phase liquid chromatography directly coupled to a linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FTICR-MS). A. flavus is a toxic filamentous fungus that significantly impacts the agricultural economy and human health. SILAC labeling improved the confidence of protein identification, and we observed 1318 unique protein masses corresponding to 659 SILAC pairs, of which 22 were confidently identified. However, we have observed some limiting issues with regard to protein quantification using top-down MS/MS analyses of SILAC-labeled proteins. The role of SILAC labeling in the presence of competing endogenously produced amino acid residues and its impact on quantification of intact species are discussed in detail.     

Imaging mass spectrometry of three-dimensional cell culture systems

Three-dimensional (3D) cell cultures have increased complexity compared to simple monolayer and suspension cultures, recapitulating the cellular architecture and molecular gradients in tissue. As such, they are popular for in vitro models in biological research. Classical imaging methodologies, like immunohistochemistry, are commonly used to examine the distribution of specific species within the spheroids. However, there is a need for an unbiased discovery-based methodology that would allow examination of protein/peptide distributions in 3D culture systems, without a need for prior knowledge of the analytes. We have developed a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS)-based imaging approach to examine protein distributions in 3D cell culture models. Using colon carcinoma cell lines, we detect changes in the spatial distribution of proteins across 3D culture structures. To identify the protein species present, we are combining results from the MS/MS capabilities of MALDI-MS to sequence peptides in a de novo fashion and nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) of homogenized cultures. As a proof-of-principle, we have identified cytochrome C and Histone H4 as two of the predominant protein species in the 3D colon carcinoma cultures.     

Analysis of complex protein mixtures with improved sequence coverage using (CE-MS/MS)n

Identification of proteins, in a complex protein mixture, using one-dimensional high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analysis of its digest, usually suffers from low sequence coverage. There are several reasons for the low coverage including undersampling, wide concentration dynamic range of the proteins in a complex protein mixture, and wide range of electrospray ionization efficiency of peptides under each mobile-phase composition. To address this low sequence coverage, we introduce a novel technique, (CE-MS/MS)n, which utilizes the most significant advantages of CE-MS/MS, including economy of sample size, fast analysis time, and high separation efficiency, to increase the sequence coverage of a complex protein mixture. Based on these characteristics, (CE-MS/MS)n can be performed in which multiple CE-MS/MS subanalyses (injections followed by analyses) are analyzed and experimental variables are manipulated during each CE-MS/MS subanalysis in order to maximize sequence coverage. (CE-MS/MS)n is a practical technique since each CE-MS/MS subanalysis consumes <10 nL, and each CE-MS/MS subanalysis takes approximately 10 min; therefore, several subanalyses can be performed in approximately 1 h consuming only nanoliters of the sample. Two techniques have been introduced to address the undersampling: (1) (CE-MS/MS)n using dynamic exclusion. In this technique, several CE-MS/MS analyses (injection followed by separation) were performed in one run using the dynamic exclusion capability of the mass spectrometer until all peptide peaks were analyzed by MS/MS. (2) Gas-phase fractionation. In this technique, (CE-MS/MS)n is performed by scanning a narrow mass range (every approximately 100 m/z) during each CE-MS/MS subanalysis without using dynamic exclusion. Under this condition, in each subanalysis, the number of peptides available for MS/MS analysis is significantly reduced, and peptides with the same nominal masses are analyzed, thereby increasing sequence coverage. Additionally, to address the lack of detection of low-level peptides in a mixture containing a wide concentration dynamic range, the concentration of the sample was systematically increased in each subanalysis (while utilizing dynamic exclusion) so that low-intensity peptides would rise above the mass spectrometer threshold and, consequently, undergo MS/MS analysis. Moreover, to alter the ionization efficiency of peptides with low electrospray ionization efficiency, and to change the migration behavior of comigrating peptides under a specific liquid composition, the CE background electrolyte was modified in several subanalyses to further improve sequence coverage. The combination of the above-mentioned techniques was applied to the analysis of the tryptic digests of three well-characterized protein mixtures: a six-protein mixture with average MW of approximately 26,000 (standard I), a six-protein mixture with an average MW approximately 49,000 (standard II), and a more complex protein mixture containing 55 proteins (E. coli ribosomal proteins). In approximately 1 h, when the MS/MS of the peptides were manually checked, all peptides that produced peaks under electrospray ionization in the scanned range of the analysis (500-2000 m/z) and within the practical fragmentation capability of the MS (peptides with MW <3500) were identified for standard I by consuming only 200 fmol of each protein. When searched against a Swissprot database, the average sequence coverage for the standard I, II, and E. coli's ribosomal proteins were 57, 34, and 15%, respectively.     

Ultratrace LC/MS proteomic analysis using 10-microm-i.d. Porous layer open tubular poly(styrene-divinylbenzene) capillary columns

In this paper, the preparation and performance of long, high-efficiency poly(styrene-divinylbenzene) (PS-DVB), 10-microm-i.d. porous layer open tubular (PLOT) capillary columns are described. PLOT capillaries ( approximately 3% RSD column-to-column retention time), with relatively high permeability, were prepared by in-situ polymerization. Relatively high loading capacities, approximately 100 fmol for angiotensin I and approximately 50 fmol for insulin, were obtained with a 4.2 m x 10-microm-i.d. PLOT column. Low detection levels (attomole to sub-attomole) were achieved when the column was coupled on-line with a linear ion trap MS (LTQ). Analysis of human epidermal growth factor receptor (EGFR), a large transmembrane tyrosine kinase receptor with heterogeneous phosphorylation and glycosylation structures, was obtained at the 25 fmol level. The PLOT column yielded a peak capacity of approximately 400 for the separation of a complex tryptic digest mixture when the sample preparation included a 50-microm-i.d. PS-DVB monolithic precolumn and ESI-MS detection. As an example of the power of the column, 3046 unique peptides covering 566 distinct Methanosarcina acetivorans proteins were identified from a 50 ng in-gel tryptic digest sample combining five cuts in a single LC/MS/MS analysis using the LTQ. The results demonstrate the potential of the PLOT column for high-resolution LC/MS at the ultratrace level.     

A proteome strategy for fractionating proteins and peptides using continuous free-flow electrophoresis coupled off-line to reversed-phase high-performance liquid chromatography

Extensive prefractionation is now considered to be a necessary prerequisite for the comprehensive analysis of complex proteomes where the dynamic range of protein abundances can vary from approximately 10(6) for cells to approximately 10(10) for tissues such as blood. Here, we describe a high-resolution 2D protein separation system that uses a continuous free-flow electrophoresis (FFE) device to fractionate complex protein mixtures by solution-phase isoelectric focusing (IEF) into 96 well-defined pools, each separated by approximately 0.02-0.10 pH unit depending on the gradient created, followed by rapid (approximately 6 min per analysis) reversed-phase high-performance liquid chromatography (RP-HPLC) of each FFE pool. Fractionated proteins are readily visualized in a virtual 2D format using software that plots protein loci, pI in the first dimension and relative hydrophobicity (i.e., RP-HPLC retention time) in the second dimension. By coupling a diode-array detector in line with a multiwavelength fluorescence detector, separated proteins can be monitored in the RP-HPLC eluent by both UV absorbance and intrinsic fluorescence simultaneously from a single experiment. Triplicate analyses of standard proteins using a pH 3-10 gradient conducted over a 3-day period revealed a high system reproducibility with a SD of 0.57 (0.05 pH unit) within the FFE pools and 0.003 (0.18 s) for protein retention times in the second-dimension RP-HPLC step. In addition, we demonstrate that the FFE-IEF/RP-HPLC separation strategy can also be applied to complex mixtures of low molecular weight compounds such as peptides. With the facile ability to measure the pH of the isoelectric focused pools, peptide pI values can be estimated and used to qualify peptide identifications made using either MS/MS sequencing approaches or pI discriminated peptide mass fingerprinting. The calculated peak capacity of this 2D liquid-based FFE-IEF/RP-HPLC system is 6720.     

Distinguishing and quantifying peptides and proteins containing D-amino acids by tandem mass spectrometry

Tandem mass spectrometry (MS/MS) utilizing both electron capture dissociation (ECD) and collisionally activated dissociation (CAD) was used to develop a qualitative and quantitative analytical method for chiral analysis of individual amino acid residues in polypeptides. ECD produced a more distinct chiral recognition than CAD, which is attributed to the smaller degree of vibrational excitation in ECD. Several peptide and protein model systems were used in this study, including the smallest known protein, tryptophan cage, a lactoferrin peptide, and the biologically relevant opioid peptide, dermorphin. An adaptation of the kinetic method was used to quantify the degree of separation between fragmentation patterns of stereoisomeric peptides as a function of fragment ion abundances. The obtained calibration scale for relative abundances of d-amino acids in diastereomeric peptide mixtures was accurate to 1% for ECD and to 3-5% for CAD. It was found that separation and quantification of stereoisomers could be advantageously performed by nanoflow reversed-phase liquid chromatography, with the objective of on-line MS/MS limited to stereoisomer identification. This technique shows promise for the analysis of chiral substitution in peptides and proteins, broadening the application area for tandem mass spectrometry.     

Sonic spray ionization technology: performance study and application to a LC/MS analysis on a monolithic silica column for heroin impurity profiling

Sonic spray (SS) ionization is a relatively novel atmospheric pressure ionization technique for LC/MS, based on the principle of "spray ionization", which only recently became commercially available. In this paper, we evaluate the performance of this ion source as an interface for LC/MS in comparison with the more traditional and better studied pneumatically assisted electrospray or ion spray (IS). The effect of organic modifiers, volatile acids, and buffer systems in the LC eluent on the ionization efficiency of both interfaces is described and some possible explanations for the observed phenomena are highlighted. We could conclude that the presence of organic solvents gradually increased the ionization efficiency for IS and SS, while volatile acids or buffers gave a significant signal suppression. Furthermore, we present the application of the sonic spray interface to a fast LC/MS analysis, for the simultaneous determination of the seven prime opium alkaloids in heroin impurity profiling. Chromatographic separation is performed in 5 min on a monolithic silica column (Chromolith Performance) with a gradient elution system and an optimized flow of 5 mL/min. By means of a postcolumn split of approximately 1/20, a coupling between the fast LC system and the mass spectrometer is made. The method is validated and successfully applied to the analysis of real-time seized heroin street samples.     

Automated 20 kpsi RPLC-MS and MS/MS with chromatographic peak capacities of 1000-1500 and capabilities in proteomics and metabolomics

Proteomics analysis based-on reversed-phase liquid chromatography (RPLC) is widely practiced; however, variations providing cutting-edge RPLC performance have generally not been adopted even though their benefits are well established. Here, we describe an automated format 20 kpsi RPLC system for proteomics and metabolomics that includes on-line coupling of micro-solid phase extraction for sample loading and allows electrospray ionization emitters to be readily replaced. The system uses 50 microm i.d. x 40-200 cm fused-silica capillaries packed with 1.4-3-microm porous C18-bonded silica particles to obtain chromatographic peak capacities of 1000-1500 for complex peptide and metabolite mixtures. This separation quality provided high-confidence identifications of >12 000 different tryptic peptides from >2000 distinct Shewanella oneidensis proteins (approximately 40% of the proteins predicted for the S. oneidensis proteome) in a single 12-h ion trap tandem mass spectrometry (MS/MS) analysis. The protein identification reproducibility approached 90% between replicate experiments. The average protein MS/MS identification rate exceeded 10 proteins/min, and 1207 proteins were identified in 120 min through assignment of 5944 different peptides. The proteomic analysis dynamic range of the 20 kpsi RPLC-ion trap MS/MS was approximately 10(6) based on analyses of a human blood plasma sample, for which 835 distinct proteins were identified with high confidence in a single 12-h run. A single run of the 20 kpsi RPLC-accurate mass MS detected >5000 different compounds from a metabolomics sample.     

Pronase-immobilized enzyme reactor: an approach for automation in glycoprotein analysis by LC/LC-ESI/MSn

An automated analytical approach is proposed for simultaneous characterization of glycan and peptide moieties in pronase-generated glycopeptides. The proposed method is based on the use of a new pronase-immobilized enzyme reactor for the on-line rapid digestion of the target glycoprotein. By coupling the bioreactor to a Hypercarb chromatographic trap column, on-line selective glycopeptide enrichment prior to normal-phase liquid chromatography-mass spectrometry was obtained. A detailed study was carried out for integration and automation of each phase of the proposed analytical procedure. On-line digestion allowed extensive cleavage of the model protein (ribonuclease B), yielding to glycopeptides with peptide moieties up to eight amino acids, carrying the Man5-Man9 N-glycans each, selectively resolved on an Amide-80 column. The use of a linear ion trap instrument resulted in efficient ion capture and led to MS3 acquisition times and spectra quality similar to those for MS2, allowing the unambiguous identification of glycan (MS2) and peptide (MS3) sequences. The proposed procedure reduces the glycoprotein analysis time from approximately 3 days, as in most of the traditional off-line methods, to approximately 1 h.     

Capillary trap column with strong cation-exchange monolith for automated shotgun proteome analysis

A 150 microm internal diameter capillary monolithic column with a strong cation-exchange stationary phase was prepared by direct in situ polymerization of ethylene glycol methacrylate phosphate and bisacrylamide in a trinary porogenic solvent consisting dimethylsulfoxide, dodecanol, and N,N'-dimethylformamide. This phosphate monolithic column exhibits higher dynamic binding capacity, faster kinetic adsorption of peptides, and more than 10 times higher permeability than the column packed with commercially available strong cation-exchange particles. It was applied as a trap column in a nanoflow liquid chromatography-tandem mass spectrometry system for automated sample injection and online multidimensional separation. It was observed that the sample could be loaded at a flow rate as high as 40 microL/min with a back pressure of approximately 1300 psi and without compromising the separation efficiency. Because of its good orthogonality to the reversed phase separation mechanism, the phosphate monolithic trap column was coupled with a reversed-phase column for online multidimensional separation of 19 microg of the tryptic digest of yeast proteins. A total of 1522 distinct proteins were identified from 5608 unique peptides (total of 54,780 peptides) at the false positive rate only 0.46%.     

Analysis of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in urine by extraction on a molecularly imprinted polymer column and liquid chromatography/atmospheric pressure ionization tandem mass spectrometry

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is present in the urine of tobacco users and, at lower concentrations, in the urine of nonsmokers exposed to secondhand smoke. NNAL is a valuable biomarker of human exposure to the carcinogenic nitrosamines in tobacco and tobacco smoke, but its presence at low concentrations in urine requires sensitive and often complex analytic procedures. In this report, we describe the development of an efficient method for the analysis of NNAL in human urine using liquid chromatography/atmospheric pressure ionization tandem mass spectrometry (LC/MS/MS) combined with a novel sample cleanup based on a molecularly imprinted polymer (MIP) column developed specifically for this assay. Our results suggest that this combination of MIP column extraction and LC/MS/MS can provide a sensitive and relatively simple analytical method suitable for application to epidemiologic investigations of health risks associated with the exposure to tobacco smoke or SHS in both smokers and nonsmokers.