Two-dimensional strong cation exchange/porous layer open tubular/mass spectrometry for ultratrace proteomic analysis using a 10 μm id poly(styrene- divinylbenzene) porous layer open tubular column with an on-line triphasic trapping column (original) (raw)
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Analytical Chemistry, 2007
Following on our recent work, on-line one dimensional (1D) and two dimensional (2D) PLOT/LC-ESI-MS platforms using 3.2 m × 10 μm i.d. poly(styrenedivinylbenzene) (PS-DVB) porous layer open tubular (PLOT) columns have been developed to provide robust, high performance and ultrasensitive proteomic analysis. Using a PicoClear tee, the dead volume connection between a 50 μm i.d. PS-DVB monolithic microSPE column and the PLOT column was minimized. The microSPE/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 to 40 kDa) of a cervical cancer (SiHa) cell line. As an example of the sensitivity of the system, ∼2.5 ng of protein in 2 μL solution, an amount corresponding to 20 SiHa cells, was subjected to on-line microSPE-PLOT/LC-ESIMS/MS analysis using a linear ion trap MS. 237 peptides associated with 163 unique proteins were identified from a single analysis when using stringent criteria associated with a false positive rate less than 1%. The number of identified peptides and proteins increased to 638 and 343, respectively, as the injection amount was raised to ∼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 cm × 75 μm 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 ∼5% of the injected sample amount. The resolving power of the microSPE/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 5 ion exchange fractions in online 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. Global characterization of proteins from complex mixtures over a wide dynamic concentration range is one of the challenges of current proteomic studies. 1 Multidimensional high performance liquid chromatography (HPLC) has received considerable attention for such analyses. 2-3 Among the several LC combinations, coupling strong cation exchange (SCX) as
Journal of Chromatography A, 2005
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 C 18 reversed-phase (RP) particles and SCX resin in a silica capillary tubing (1.5 cm × 200 m I.D. for SCX, 0.7 cm × 200 m 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 L of NH 4 HCO 3 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 g 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.
ELECTROPHORESIS, 2002
The present report describes the design and application of a dual sprayer system for high-throughput proteome analysis. This system comprises parallel solid-phase extraction cartridges used for preconcentration and desalting of proteolytic digests prior to nanoelectrospray mass spectrometry analyses. Tryptic peptides from in-gel digest of protein bands/spots are first adsorbed on styrene divinyl benzene membrane and subsequently eluted with a short plug of organic buffer prior to infusion to the mass spectrometer at a flow rate of typically 500 nL/min. Tryptic peptide eluting from the membrane are analyzed by the mass spectrometer by moving in turn each sprayer in front of the sampling orifice. Sequential injection, preconcentration and analyses of tryptic digests are typically achieved with a throughput of up to 3.5 min/sample and a detection limit of approximately 8-80 fmol per injection. Replicate injections of peptide mixtures indicated that reproducibility of peak areas ranged from relative standard deviations (RSD) of 1.1% to 4.5%. The application of this device is demonstrated for digests of gel-isolated proteins obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of rat liver plasma membrane and from two-dimensional gel electrophoresis of total cell lysate extracts from human prostatic cancer cell**.
Journal of Chromatography A, 2007
This paper describes approaches to optimize the chromatographic performance for our recently developed LC-MS platform, extended range proteomic analysis (ERPA), for comprehensive protein characterization at the ultratrace level. Large digested peptide fragments up to 10 kDa (e.g. from lysyl endopeptidase digestion) with or without modifications were well separated with high resolution using narrow bore (20 and 50 μm I.D.) poly(styrene-divinylbenzene) (PS-DVB) monolithic columns constructed by in situ solution polymerization. Importantly, the macroporous structure of the monolithic columns facilitated mass transport of large peptides with improved recovery relative to small pore size reversed-phase packings. High sequence coverage (>95 %), including identification of phosphorylated and glycosylated particles was achieved for β-casein and epidermal growth factor receptor (EGFR) at the 4 fmol and 20 fmol levels per injection, respectively, using the 20 μm I.D. PS-DVB monolithic column. For peptides with greater ionization efficiency, the detection limit could be lowered to ~ 400 zmol. Typically, the separation system produced a peak capacity of ~200 for a 10 cm column. This paper demonstrates that narrow-bore monolithic columns are suitable for high sensitivity and high resolution separation of large peptide fragments by LC-MS analysis.
Journal of Proteome Research, 2008
Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. Analysis platforms must balance sensitivity for peptide detection, reproducibility of detected peptide inventories and analytical throughput for protein amounts commonly present in tissue biospecimens (<100 µg), such that platform stability is sufficient to detect modest changes in complex proteomes. We compared shotgun proteomics platforms by analyzing tryptic digests of whole cell and tissue proteomes using strong cation exchange (SCX) and isoelectric focusing (IEF) separations of peptides prior to LC-MS/MS analysis on a LTQ-Orbitrap hybrid instrument. IEF separations provided superior reproducibility and resolution for peptide fractionation from samples corresponding to both large (100 µg) and small (10 µg) protein inputs. SCX generated more peptide and protein identifications than did IEF with small (10 µg) samples, whereas the two platforms yielded similar numbers of identifications with large (100 µg) samples. In nine replicate analyses of tryptic peptides from 50 µg colon adenocarcinoma protein, overlap in protein detection by the two platforms was 77% of all proteins detected by both methods combined. IEF more quickly approached maximal detection, with 90% of IEF-detectable medium abundance proteins (those detected with a total of 3-4 peptides) detected within three replicate analyses. In contrast, the SCX platform required six replicates to detect 90% of SCX-detectable medium abundance proteins. High reproducibility and efficient resolution of IEF peptide separations make the IEF platform superior to the SCX platform for biomarker discovery via shotgun proteomic analyses of tissue specimens.
Analytical Chemistry, 2005
A novel integrated multidimensional liquid chromatography (IMDL) method is demonstrated for the separation of peptide mixtures by two-dimensional HPLC coupled with ion trap mass spectrometry. The method uses an integrated column, containing both strong cation exchange and reversed-phase sections for two-dimensional liquid chromatography. The peptide mixture was fractionated by a pH step using a series of pH buffers, followed by reversed-phase chromatography. Since no salt was used during separation, the integrated multidimensional liquid chromatography can be directly connected to mass spectrometry for peptide analysis. The pH buffers were injected from an autosampler, and the entire process can be carried out on a one-dimensional liquid chromatography system. In a single analysis, the IMDL system, coupled with linear ion trap mass spectrometry, identified more than 2000 proteins in mouse liver. The peptides were eluted according to their pI distribution. The resolution of the pH fractionation is ∼0.5 pH unit. The method has low overlapping across pH fractions, good resolution of peptide mixture, and good correlation of peptide pIs with pH steps. This method provides a technique for largescale protein identification using existing one-dimensional HPLC systems.
Journal of Chromatography A, 2007
Nanoliter flow rate is optimum for separation in capillary column liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). In order to develop a high-performance automated proteome analysis system allowing direct injection sample containing detergents, the influence of void volume varied from 0 to 5 L on the separation performance and proteomic coverage of sample injection system using strong cation-exchange (SCX) trap column was investigated, it was found the void volume hardly affects the separation performance by using SCX trap column. Thus, a fully automated sample injection system using SCX trap column and ten-port switching valve was established for efficient shotgun proteome analysis. In this system, a nanoflow switching valve and a microtee were used to connect the SCX trap and analytical columns, and the uncleaned samples of proteolytic digests containing contaminants could be directly injected with minor influence on the separation performance, which was demonstrated to be a useful strategy in proteome analysis.
Analytical Chemistry, 2005
We describe the preparation and performance of highefficiency 70 cm × 20 μm i.d. silica-based monolithic capillary LC columns. The monolithic columns at a mobile-phase pressure of 5000 psi provide flow rates of ∼40 nL/min at a linear velocity of ∼0.24 cm/s. The columns provide a separation peak capacity of ∼420 in conjunction with both on-line coupling with microsolidphase extraction and nanoelectrospray ionization-mass spectrometry. Performance was evaluated using a Shewanella oneidensis tryptic digest, and ∼15-amol detection limits for peptides were obtained using a conventional ion trap and MS/MS for peptide identification. The sensitivity and separation efficiency enabled the identification of 2367 different peptides covering 855 distinct S. oneidensis proteins from a 2.5-μg tryptic digest sample in a single 10-h analysis. The number of identified peptides and proteins approximately doubled when the effective separation time was extended from 200 to 600 min. The number of identified peptides increased from 32 to 390 as the injection amount was increased from 0.5 to 100 ng. Both the run-to-run and column-to-column reproducibility for proteomic analyses were also evaluated.
On-line strong cation exchange μ-HPLC-ESI-MS/MS for protein identification and process optimization
Journal of the American Society for Mass Spectrometry, 2003
We have developed an on-line strong cation exchange (SCX)-ESI-MS/MS platform for the rapid identification of proteins contained in mixtures. This platform consists of a SCX precolumn followed by a nanoflow SCX column on-line with an electrospray ion trap mass spectrometer. We also used this platform to study the dynamics of peptide separation/ extraction by SCX, in particular to understand the parameters affecting the performance of SCX in multidimensional chromatography. For example, we have demonstrated that the buffer typically used for tryptic digestion of protein mixtures can have a detrimental effect on the chromatographic behaviour of peptides during SCX separations, thereby affecting certain peptide quantitation approaches that rely on reproducible peptide fractionation. We have also demonstrated that band broadening results when a step (discontinuous) gradient approach is used to displace peptides from the SCX precolumn, reducing the separation power of SCX in multidimensional chromatography. In contrast, excellent chromatographic peak shapes are observed when a defined (continuous) gradient is used. Finally, using a tryptic digest of a protein extract derived from human K562 cells, we observed that larger molecular weight peptides are identified using this on-line SCX approach compared to the more conventional reverse phase (RP) LC/MS approach. Both methods used in tandem complement each other and can lead to a greater number of peptide identifications from a given sample. P rotein expression is a highly regulated process involving multiple genes. Their levels of expression can be regulated at the level of mRNA expression, through degradation pathways, through post-translational modification/processing, through localization, and through secretion. As demonstrated [1], there is not always a clear relationship between the level of mRNA expression and protein expression. Thus, greater insight into protein expression is key to understanding biological processes and diseases. Unfortunately, the equivalent of the genomic gene chip technology is not available for the rapid expression analysis of proteins. Protein expression profiles have been mainly obtained through the combination of 2-D gel electrophoresis and mass spectrometry. Although 2-D gel electrophoresis is a powerful protein separation technique, its combination with mass spectrometry does not always deliver the appropriate sensitivity for the discovery of low-level proteins . In addition, 2-D gel analysis can be biased against the detection of membrane proteins because of solubility issues. Al-though many efforts have led to improved processing of various sample types including membrane proteins by 2-D gel analysis, several challenges continue to remain . Finally and in spite of the tremendous utility of 2-D gels, the technical challenges associated with this technique are limiting its widespread utilization and applicability.