High-throughput peptide identification from protein digests using data-dependent multiplexed tandem FTICR mass spectrometry coupled with capillary liquid chromatography

Tandem mass spectrometry (MS/MS) plays an important role in the unambiguous identification and structural elucidation of biomolecules. In contrast to conventional MS/MS approaches for protein identification where an individual polypeptide is sequentially selected and dissociated, a multiplexed-MS/MS approach increases throughput by selecting several peptides for simultaneous dissociation using either infrared multiphoton dissociation (IRMPD) or multiple frequency sustained off-resonance irradiation (SORI) collisionally induced dissociation (CID). The high mass measurement accuracy and resolution of FTICR combined with knowledge of peptide dissociation pathways allows the fragments arising from several different parent ions to be assigned. Herein we report the application of multiplexed-MS/MS coupled with on-line separations for the identification of peptides present in complex mixtures (i.e., whole cell lysate digests). Software was developed to enable "on-the-fly" data-dependent peak selection of a subset of polypeptides from each FTICR MS acquisition. In the subsequent MS/MS acquisitions, several coeluting peptides were fragmented simultaneously using either IRMPD or SORI-CID techniques. The utility of this approach has been demonstrated using a bovine serum albumin tryptic digest separated by capillary LC where multiple peptides were readily identified in single MS/MS acquisitions. We also present initial results from multiplexed-MS/MS analysis of a D. radiodurans whole cell digest to illustrate the utility of this approach for high-throughput analysis of a bacterial proteome.     

Use of a single-quadrupole mass spectrometer for collision-induced dissociation studies of multiply charged peptide ions produced by electrospray ionization.

The feasibility of obtaining the collision-induced dissociation (CID) spectra of multiply charged peptide ions produced by electrospray ionization in a simple and inexpensive single-quadrupole mass spectrometer is demonstrated. Collisional activation was carried out in the high-pressure region between the capillary exit and the skimmer entrance to the mass analyzer. The CID of multiply charged peptide ions is very efficient, and the observed fragment ion intensities are typically 1-5% of the parent ion intensity prior to CID. About 70 pmol of the peptide is consumed in obtaining each CID spectrum. Spectra obtained by CID of multiply charged ions from bradykinin, angiotensin II, two peptides with features similar to tryptic peptides, and a synthetic analogue of a component of TGF-alpha containing two disulfide bonds are shown. The influence of the primary structure of the peptide on the observed fragmentation pathways is discussed. Although the present single-quadrupole configuration is simple and effective, the inability to choose a particular parent ion for collisional activation makes it less powerful than the triple-quadrupole configuration for mixtures of peptides and peptide samples that yield more than one charge state in the normal mass spectrum. However, it has the potential for inexpensively obtaining sequence information of proteins at high sensitivity by analyzing the pure tryptic peptides obtained by on-line or off-line chromatographic separation of tryptic digests.     

Analysis of histidine phosphorylation using tandem MS and ion-electron reactions

Phosphorylation of proteins is essential in intracellular signal transduction pathways in eukaryotic and prokaryotic cells. Histidine phosphorylation plays an important role in two-component signal transduction in bacteria. In this study, we describe the characterization of a synthetic histidine-phosphorylated peptide with four different mass spectrometric (MS) fragmentation techniques: Collision-induced dissociation (CID), electron capture dissociation, electron-transfer dissociation, and electron detachment dissociation. Furthermore, LC-MS methods were developed to detect histidine-phosphorylated peptides, which are acid-labile, in more complex samples. From these results, we concluded that nonacidic solvent systems or fast LC methods provide the best conditions for separation of histidine-phosphorylated peptides prior to electrospray ionization mass spectrometry analysis. Electron-based fragmentation methods should be used for determination of histidine phosphorylation sites, since CID results in very facile phosphate-related neutral losses. The developed LC-MS/MS methods were successfully applied to a tryptic digest of the cytoplasmic part of the histidine kinase EnvZ, which was in vitro autophosphorylated. Finally, a new method is described for nonretentive solid-phase extraction of histidine-phosphorylated peptides using polymeric Strata-X microcolumns.     

Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI fourier transform ion cyclotron resonance mass spectrometry

Subfemtomole peptide sequence analysis has been achieved using microcapillary HPLC columns, with integrated nanoelectrospray emitters, coupled directly to a Fourier transform ion cyclotron resonance mass spectrometer. Accurate mass (+/-0.010 Da) peptide maps are generated from a standard six-protein digest mixture, whose principle components span a concentration dynamic range of 1000:1. Iterative searches against approximately 189000 entries in the OWL database readily identify each protein, with high sequence coverage (20-60%), from as little as 10 amol loaded on-column. In addition, a simple variable-flow HPLC apparatus provides for on-line tandem mass spectrometric analysis of tryptic peptides at the 400-amol level. MS/MS data are searched against approximately 280000 entries in a nonredundant protein database using SEQUEST. Accurate precursor and product ion mass information readily identifies primary amino acid sequences differing by asparagine vs aspartic acid (deltam = 0.98 Da) and glutamine vs lysine (deltam = 0.036 Da).     

Quantitative multiple reaction monitoring of peptide abundance introduced via a capillary zone electrophoresis-electrospray interface

We demonstrate the use of capillary zone electrophoresis with an electrokinetic sheath-flow electrospray interface coupled to a triple-quadrupole mass spectrometer for the accurate and precise quantification of Leu-enkephalin in a complex mixture using multiple-reaction monitoring (MRM). Assay time is <6 min, with no re-equilibration required between runs. A standard curve of Leu-enkephalin was performed in the presence of a background tryptic digest of bovine albumin. We demonstrate reasonably reproducible peak heights (21% relative standard deviation), retention times (better than 1% relative standard deviation), and robust electrospray quality. Our limit of detection (3σ) was 60 pM, which corresponds to the injection of 335 zmol of peptide. This is a 10-20-fold improvement in mass sensitivity than we have obtained by nano HPLC/MRM and substantially better than reported for LC/MS/MS. Further quantification was performed in the presence of stable-isotope-labeled versions of the peptides; under these conditions, linearity was observed across nearly 4 orders of magnitude. The concentration detection limit was 240 pM for the stable-isotope-labeled quantification.     

Primary structure determination of peptides and enzymatically digested proteins using capillary liquid chromatography/mass spectrometry and rapid linked-scan techniques

Primary protein sequences were determined for both peptides and enzymatically digested proteins by rapid linked-scan (B/E) liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) at the low-picomole level (10-50 pmol). During the course of a single LC/MS/MS analysis, we demonstrated that it is possible to generate interpretable collision-induced dissociation spectra of the eluting proteolytic peptides. Molecular weights of tryptic peptides were established by using 1/10 of the protein digest by operating in the capillary LC/frit-FABMS mode. Peptides exhibiting the strongest MH+ ions were then selected for subsequent LC/MS/MS analysis (typically 1/5 of the remaining protein digest). Elution times for each chromatographic peak were generally greater than 30 s. It was therefore possible to obtain a minimum of six B/E fast linked-scan spectra during the course of elution of each peptide component. Typically, B/E linked scans of the greatest ion abundance (obtained at the chromatographic peak maximum) were averaged to enhance the signal/noise ratio at these low-picomole levels. Unit resolution was observed for product ions below m/z 1000. Rapid linked scanning by LC/frit-FABMS/MS provided mass assignments for product ions within 0.2-0.3 amu of theoretical values. Side-chain fragment ions (wn and dn) were also observed, which allowed for the differentiation of isobaric amino acids (e.g., leucine and isoleucine). Examples of the application of this fast linked-scan technique to LC/MS/MS are presented for complex mixtures of unknown peptides and the tryptic digestion of phosphorylated beta-casein.     

Chip-based capillary electrophoresis/mass spectrometry determination of carnitines in human urine

A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system is described for the CE separation and on-line electrospray detection of carnitine and selected acylcarnitines from mixtures of analytical standards as well as extracts of fortified human urine. Chip-based CE/MS experiments in two different laboratories were carried out using a triple-quadrupole mass spectrometer and a quadrupole time-of-flight (QTOF) mass spectrometer, respectively. The glass chips used with both systems were comparably equipped with a microfabricated capillary electrophoresis (CE) channel but with different electrosprayers. The quadrupole chip-based CE/MS experiments employed a miniature coupled microsprayer, which allowed coupling of the microelectrospray process via a micro liquid junction at the exit of the CE capillary channel. Selected ion monitoring (SIM) CE/MS experiments were employed for all of the quadrupole CE/MS work. The QTOF CE/MS full-scan single MS and MS/MS experiments were carried out in another laboratory using accurate mass measurement TOF mass spectrometry techniques. The electrospray process that was employed with the QTOF system differed in that an inserted nanoelectrospray capillary needle was carefully affixed into a flat-bottomed hole that was aligned with the CE channel exit orifice. SIM CE/MS using the described quadrupole system provided acceptable ion current electropherograms from fmole levels from analytical standard solutions of carnitine and acylcarnitines that were manually injected (loaded) onto the chip. In addition, the corresponding electropherograms for human urine fortified with the target carnitine and acylcarnitines at a 10-20 microg/mL (35-124 microM) level were obtained via SIM CE/MS techniques. The measured CE separation efficiency for the SIM CE/MS electropherograms was determined to be 2860 plates (peak width at half-height method or N = 5.54(T/WO.5(2)), and carnitine and three acylcarnitines were separated in less than 48 s. In contrast, using quadrupole-TOF technologies, the same samples could be diluted by a factor of 2-4 to obtain a comparable detector response for the target compounds. In the full-scan, single mass analyzer mode (m/z 150-500), the CE separation efficiency was measured to be 2600 plates, but mass measurement accuracy was less than 5.0 ppm for the quaternary cations. In the CE/MS/MS mode, full-scan collision-induced dissociation (CID) mass spectra were obtained with a mass accuracy of < or =10 ppm for the higher mass ions and < or =27 ppm for the lower mass product ions. These results demonstrate the feasibility for on-chip CE separation and electrospray mass spectrometric detection for these important compounds in synthetic mixtures, as well as in human urine extracts.     

Tandem parallel fragmentation of peptides for mass spectrometry

Parallel fragmentations of peptides in the source region and in the collision cell of tandem mass spectrometers are sequentially combined to develop parallel collision-induced-dissociation mass spectrometry (p2CID MS). Compared to MS/MS spectra, the p2CID mass spectra show increased signal intensities (2-400-fold) and number of sequence ions. This improvement is attributed to the fact that p2CID MS virtually samples all the ions generated by electrospray ionization, including intact and fragment ions of different charge states from a peptide. We implement the method using a quadrupole time-of-flight tandem mass spectrometer. The instrument is operated in TOF-MS mode that allows the ions from source region broadband-passing the first mass analyzer to enter the collision cell. Cone voltage and collision energy are investigated to optimize the outcome of the two parallel CID processes. In the in-source parallel CID, elevated cone voltage produces singly charged intact peptide ions and large fragment ions, as well as decreases the charge-state distribution of peptide ions mainly to double and single charges. The in-collision-cell parallel CID is optimized to dissociate the ions from the source region to produce small and medium fragment ions. The method of p2CID MS is especially useful for sequencing of large peptides with labile amide bonds and peptides with C-terminal arginine. It has unique potential for de novo sequencing of peptides and proteome analysis, especially for affinity-enriched subproteomes.     

Preparation of 20-microm-i.d. silica-based monolithic columns and their performance for proteomics analyses

We describe the preparation and performance of high-efficiency 70 cm x 20 microm i.d. silica-based monolithic capillary LC columns. The monolithic columns at a mobile-phase pressure of 5000 psi provide flow rates of approximately 40 nL/min at a linear velocity of approximately 0.24 cm/s. The columns provide a separation peak capacity of approximately 420 in conjunction with both on-line coupling with microsolid-phase extraction and nanoelectrospray ionization-mass spectrometry. Performance was evaluated using a Shewanella oneidensis tryptic digest, and approximately 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-microg 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.     

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.     

Very high pressure gradient LC/MS/MS

A very high pressure liquid chromatography (VHPLC) system was constructed by modifying a commercially available pump in order to achieve pressures in excess of 1,200 bar (17,500 psi). A computer-controlled low-pressure mixer was used to generate solvent gradients. Protein digests were rapidly analyzed by reversed-phase VHPLC with linear solvent gradients coupled to either a tandem mass spectrometer using electrospray ionization or a UV/visible detector. The separations were performed at pressures ranging from 790 (11,500 psi) to 930 bar (13,500 psi) in 22-cm-long capillary columns packed with C18-modified 1.5-microm nonporous silica particles. A digest of bovine serum albumin (BSA) was analyzed by the VHPLC system connected to a mass spectrometer in MS mode. An analysis of 12.5 fmol of sample gave signal-to-noise ratios of tryptic peaks greater than 10:1 in the base peak plot mass chromatogram. This system was also used to analyze a proteolytic digest of a rat liver protein excised from a 2-D gel separation of a liver tissue lysate. For this analysis, the mass spectrometer was set up to perform data-dependent scanning (automated switching from MS mode to MS/MS mode when a peak was detected) for peptide sequencing and protein identification by database searching. The results of this analysis are compared to an analysis performed on the same sample using the nanoelectrospray-MS/MS technique. Though both techniques were able to identify the unknown protein, the VHPLC method gave twice as many sequenced peptides as nanoelectrospray and improved the signal-to-noise ratio of the spectra by at least a factor of 10. Direct comparisons with nanoelectrospray for MS and MS/MS data acquisition from a BSA digest were made. These comparisons show enhancements of greater than 20-fold for VHPLC over nanoelectrospray. In addition, the VHPLC/MS/MS data acquisition was accomplished in an automated manner.     

Inlet ionization: a new highly sensitive approach for liquid chromatography/mass spectrometry of small and large molecules

Inlet ionization is a new approach for ionizing both small and large molecules in solids or liquid solvents with high sensitivity. The utility of solvent based inlet ionization mass spectrometry (MS) as a method for analysis of volatile and nonvolatile compounds eluting from a liquid chromatography (LC) column is demonstrated. This new LC/MS approach uses reverse phase solvent systems common to electrospray ionization MS. The first LC/MS analyses using this novel approach produced sharp chromatographic peaks and good quality full mass range mass spectra for over 25 peptides from injection of only 1 pmol of a tryptic digest of bovine serum albumin using an eluent flow rate of 55 μL min(-1). Similarly, full acquisition LC/MS/MS of the MH(+) ion of the drug clozapine, using the same solvent flow rate, produced a signal-to-noise ratio of 54 for the major fragment ion with injection of only 1 μL of a 2 ppb solution. LC/MS results were acquired on two different manufacturer's mass spectrometers using a Waters Corporation NanoAcquity liquid chromatograph.     

Fully automated four-column capillary LC-MS system for maximizing throughput in proteomic analyses

We describe a four-column, high-pressure capillary liquid chromatography (LC) system for robust, high-throughput liquid chromatography-mass spectrometry (LC-MS(/MS)) analyses. This system performs multiple LC separations in parallel, but staggers each of them such that the data-rich region of each separation is sampled sequentially. By allowing nearly continuous data acquisition, this design maximizes the use of the mass spectrometer. Each analytical column is connected to a corresponding ESI emitter in order to avoid the use of postcolumn switching and associated dead volume issues. Encoding translation stages are employed to sequentially position the emitters at the MS inlet. The high reproducibility of this system is demonstrated using consecutive analyses of global tryptic digest of the microbe Shewanella oneidensis.     

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry as an alternative proteomics platform to ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry for samples of intermediate complexity

We demonstrate the use of capillary zone electrophoresis with an electrokinetically pumped sheath-flow electrospray interface for the analysis of a tryptic digest of a sample of intermediate protein complexity, the secreted protein fraction of Mycobacterium marinum. For electrophoretic analysis, 11 fractions were generated from the sample using reverse-phase liquid chromatography; each fraction was analyzed by CZE-ESI-MS/MS, and 334 peptides corresponding to 140 proteins were identified in 165 min of mass spectrometer time at 95% confidence (FDR < 0.15%). In comparison, 388 peptides corresponding to 134 proteins were identified in 180 min of mass spectrometer time by triplicate UPLC-ESI-MS/MS analyses, each using 250 ng of the unfractionated peptide mixture, at 95% confidence (FDR < 0.15%). Overall, 62% of peptides identified in CZE-ESI-MS/MS and 67% in UPLC-ESI-MS/MS were unique. CZE-ESI-MS/MS favored basic and hydrophilic peptides with low molecular masses. Combining the two data sets increased the number of unique peptides by 53%. Our approach identified more than twice as many proteins as the previous record for capillary electrophoresis proteome analysis. CE-ESI-MS/MS is a useful tool for the analysis of proteome samples of intermediate complexity.     

Ultrahigh-throughput proteomics using fast RPLC separations with ESI-MS/MS

We describe approaches for proteomics analysis using electrospray ionization-tandem mass spectrometry coupled with fast reversed-phase liquid chromatography (RPLC) separations. The RPLC separations used 50-microm-i.d. fused-silica capillaries packed with submicrometer-sized C18-bonded porous silica particles and achieved peak capacities of 130-420 for analytes from proteome tryptic digests. When these separations were combined with linear ion trap tandem mass spectrometry measurements, approximately 1000 proteins could be identified in 50 min from approximately 4000 identified tryptic peptides; approximately 550 proteins in 20 min from approximately 1800 peptides; and approximately 250 proteins in 8 min from approximately 700 peptides for a S. oneidensis tryptic digest. The dynamic range for protein identification with the fast separations was determined to be approximately 3-4 orders of magnitude of relative protein abundance on the basis of known proteins in human blood plasma analyses. We found that 55% of the MS/MS spectra acquired during the entire analysis (and up to 100% of the MS/MS spectra acquired from the most data-rich zone) provided sufficient quality for identifying peptides. The results confirm that such analyses using very fast (minutes) RPLC separations based on columns packed with microsized porous particles are primarily limited by the MS/MS analysis speed.     

On-line capillary electrophoresis/microelectrospray ionization-tandem mass spectrometry using an ion trap storage/time-of-flight mass spectrometer with SWIFT technology.

The development of a system capable of the speed required for on-line capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) of tryptic digests is described. The ion trap storage/reflectron time-of-flight (IT/reTOF) mass spectrometer is used as a nonscanning detector for rapid CE separation, where the peptides are ionized on-line using electrospray ionization (ESI). The ESI produced ions are stored in the ion trap and dc pulse injected into the reTOF-MS at a rate sufficient to maintain the separation achieved by CE. Using methodology generated by software and hardware developed in our lab, we can produce SWIFT (Stored Waveform Inverse Fourier Transform) ion isolation and TICKLE activation/fragmentation voltage waveforms to generate MS/MS at a rate as high as 10 Hz so that the MS/MS spectra can be optimized on even a 1-2 s eluting peak. In CE separations performed on tryptic digests of dogfish myelin basic protein (MBP) where eluting peaks 4-8 s wide are observed, it is demonstrated that an acquisition rate of 4 Hz provides > 20 spectra/peak and is more than sufficient to provide optimized MS/MS spectra of each of the eluting peaks in the electropherogram. The detailed structural analysis of dogfish MBP including several posttranslational modifications using CE-MS and CE-MS/MS is demonstrated using this method with < 10 fmol of material consumed.     

Use of peptide retention time prediction for protein identification by off-line reversed-phase HPLC-MALDI MS/MS

A new algorithm, sequence-specific retention calculator, was developed to predict retention time of tryptic peptides during RP HPLC fractionation on C18, 300-A pore size columns. Correlations of up to approximately 0.98 R2 value were obtained for a test library of approximately 2000 peptides and approximately 0.95-0.97 for a variety of real samples. The algorithm was applied in conjunction with an exclusion protocol based on mass (15 ppm tolerance) and retention time (2-min tolerance for 0.66% acetonitrile/min gradient), MART criteria to significantly reduce the instrument time required for complete MS/MS analysis of a digest separated by RP HPLC. This was confirmed by reanalyzing the set of HPLC-MALDI MS/MS data with no loss in protein identifications, despite the number of virtually executed MS/MS analyses being decreased by 57%.     

Enabling MALDI-FTICR-MS/MS for high-performance proteomics through combination of infrared and collisional activation

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a central tool for proteomic analysis, yet the singly protonated tryptic peptide ions produced by MALDI are significantly more difficult to dissociate for tandem mass spectrometry (MS/MS) than the corresponding multiply protonated ions. In order to overcome this limitation, current proteomic approaches using MALDI-MS/MS involve high-energy collision-induced dissociation (CID). Unfortunately, the use of high-energy CID complicates product ion spectra with a significant proportion of irrelevant fragments while also reducing mass accuracy and mass resolution. In order to address the lack of a high-resolution, high mass accuracy MALDI-MS/MS platform for proteomics, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and a recently developed MS/MS technique termed CIRCA (for combination of infrared and collisional activation) have been applied to proteomic analysis. Here, CIRCA is shown to be suitable for dissociating singly protonated tryptic peptides, providing greater sequence coverage than either CID or infrared multiphoton dissociation (IRMPD) alone. Furthermore, the CIRCA fragmentation spectra are of sufficient quality to allow protein identification based on the MS/MS spectra alone or in concert with the peptide mass fingerprint (PMF). This is accomplished without compromising mass accuracy or mass resolution. As a result, CIRCA serves to enable MALDI-FTICR-MS/MS for high-performance proteomics experiments.     

Trace analysis of proteins using postseparation solution-phase digestion and electrospray mass spectrometric detection of marker peptides

Analytical methodologies for the absolute quantitation of proteins typically include a digest step often using trypsin as the proteolytic enzyme. In the majority of cases, off-line and on-line digestion methods are implemented prior to an LC-MS analysis system, requiring a high sequence coverage for unambiguous protein identification. For proteins with a strong overlap in amino acid sequence, e.g., therapeutic proteins and their metabolites, it is essential to separate proteins prior to digestion and the subsequent electrospray mass spectrometry analysis of marker peptides. Here, we present an on-line postcolumn solution-phase digestion methodology that is based on the continuous infusion of the proteolytic enzyme pepsin downstream to the nano C18 reversed-phase column. Proteins are identified based on their retention time in combination with the detection of specific marker peptides formed in the postcolumn digest. The optimization of important parameters such as enzyme concentration, reaction time, and organic modifier concentration is described. We demonstrated that the continuous-flow solution-phase digest method can be coupled on-line to the reversed-phase gradient liquid chromatography separation of proteins. Detection limits obtained for five model proteins, detected as specific marker peptides with m/z values of 300-1000, range from 30 to 90 fmol, with a linear response up to 3 pmol.     

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.