Integration of microfabricated devices to capillary electrophoresis-electrospray mass spectrometry using a low dead volume connection: application to rapid analyses of proteolytic digests.

This report describes the development of a compact and versatile, micromachined chip device enabling the efficient coupling of capillary electrophoresis to electrospray mass spectrometry (CE-ESMS). On-chip separation provides a convenient means of achieving rapid sample cleanup and resolution of multicomponent samples (typically 2-5 min) prior to mass spectral analysis. A low dead volume connection facilitating the coupling of microfabricated devices to CE-ESMS was evaluated using two different interfaces. The first configuration used disposable nanoelectrospray emitters directly coupled to the chip device via this low dead volume junction, thereby providing rapid separation of complex protein digests. The performance of this interface was compared with that of more traditional configurations using a sheath flow CE-ESMS arrangement where a fused-silica capillary of varying length enabled further temporal resolution of the multicomponent samples. The sensitivity and analytical characteristics of these interfaces were investigated in both negative and positive ion modes using standard peptide mixtures. The separation performance for synthetic peptides using a chip coated with amine reagent ranged from 26,000 to 58,000 theoretical plates for a sheath flow CE-ESMS interface comprising a 15-cm CE column. Replicate injections of a dilution series of peptide standards provided detection limits of 45-400 nM without the use of on-line preconcentration devices. The reproducibility of migration time ranged from 0.9 to 1.5% RSD whereas RSDs of 5-10% were observed on peak areas. The application of these devices for the analysis of protein digests was further evaluated using on-line tandem mass spectrometry.     

Improvement in peptide detection for proteomics analyses using NanoLC-MS and high-field asymmetry waveform ion mobility mass spectrometry

Sensitive and selective detection of multiply charged peptide ions from complex tryptic digests was achieved using high-field asymmetric waveform ion mobility spectrometry (FAIMS) combined with nanoscale liquid chromatography-mass spectrometry (nanoLC-FAIMS-MS). The combination of FAIMS provided a marked advantage over conventional nanoLC-MS experiments by reducing the extent of chemical noise associated with singly charged ions and enhancing the overall population of detectable tryptic peptides. Such advantages were evidenced by a 6-12-fold improvement in signal-to-noise ratio measurements for a wide range of multiply charged peptide ions. An increase of 20% in the number of detected peptides compared to conventional nanoelectrospray was achieved by transmitting ions of different mobilities at high electric field vs low field while simultaneously recording each ion population in separate mass spectrometry acquisition channels. This method provided excellent reproducibility across replicate nanoLC-FAIMS-MS runs with more than 90% of all detected peptide ions showing less than 30% variation in intensity. The application of this technique in the context of proteomics research is demonstrated for the identification of trace-level proteins showing differential expression in U937 monocyte cell extracts following incubation with phorbol ester.     

Quantitative proteomic analysis using a MALDI quadrupole time-of-flight mass spectrometer

We describe an approach to the quantitative analysis of complex protein mixtures using a MALDI quadrupole time-of-flight (MALDI QqTOF) mass spectrometer and isotope coded affinity tag reagents (Gygi, S. P.; et al. Nat. Biotechnol. 1999, 17, 994-9.). Proteins in mixtures are first labeled on cysteinyl residues using an isotope coded affinity tag reagent, the proteins are enzymatically digested, and the labeled peptides are purified using a multidimensional separation procedure, with the last step being the elution of the labeled peptides from a microcapillary reversed-phase liquid chromatography column directly onto a MALDI sample target. After addition of matrix, the sample spots are analyzed using a MALDI QqTOF mass spectrometer, by first obtaining a mass spectrum of the peptides in each sample spot in order to quantify the ratio of abundance of pairs of isotopically tagged peptides, followed by tandem mass spectrometric analysis to ascertain the sequence of selected peptides for protein identification. The effectiveness of this approach is demonstrated in the quantification and identification of peptides from a control mixture of proteins of known relative concentrations and also in the comparative analysis of protein expression in Saccharomyces cerevisiae grown on two different carbon sources.     

MALDI quadrupole time-of-flight mass spectrometry: a powerful tool for proteomic research

A MALDI QqTOF mass spectrometer has been used to identify proteins separated by one-dimensional or two-dimensional gel electrophoresis at the femtomole level. The high mass resolution and the high mass accuracy of this instrument in both MS and MS/MS modes allow identification of a protein either by peptide mass fingerprinting of the protein digest or from tandem mass spectra acquired by collision-induced dissociation of individual peptide precursors. A peptide mass map of the digest and tandem mass spectra of multiple peptide precursor ions can be acquired from the same sample in the course of a single experiment. Database searching and acquisition of MS and MS/MS spectra can be combined in an interactive fashion, increasing the information value of the analytical data. The approach has demonstrated its usefulness in the comprehensive characterization of protein in-gel digests, in the dissection of complex protein mixtures, and in sequencing of a low molecular weight integral membrane protein. Proteins can be identified in all types of sequence databases, including an EST database. Thus, MALDI QqTOF mass spectrometry promises to have remarkable potential for advancing proteomic research.     

Direct MALDI-MS/MS of phosphopeptides affinity-bound to immobilized metal ion affinity chromatography beads

Immobilized metal ion affinity chromatography (IMAC) is a useful method to selectively isolate and enrich phosphopeptides from a peptide mixture. Mass spectrometry is a very suitable method for exact molecular weight determination of IMAC-isolated phosphopeptides, due to its inherent high sensitivity. Even exact molecular weight determination, however, is not sufficient for identification of the phosphorylation site if more than one potential phosphorylation site is present on a peptide. The previous method of choice for sequencing the affinity-bound peptides was electrospray tandem mass spectrometry (ESI-MS/MS). This method required elution and salt removal prior to MS analysis of the peptides, which can lead to sample loss. Using a matrix-assisted laser desorption/ionization (MALDI) source coupled to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer with true MS/MS capabilities, direct sequencing of IMAC-enriched peptides has been performed on IMAC beads applied directly to the MALDI target. The utility of this new method has been demonstrated on a protein with unknown phosphorylation sites, where direct MALDI-MS/MS of the tryptic peptides bound to the IMAC beads resulted in the identification of two novel phosphopeptides. Using this technique, the phosphorylation site determination is unambiguous, even with a peptide containing four potentially phosphorylated residues. Direct analysis of phosphorylated peptides on IMAC beads does not adversely affect the high-mass accuracy of an orthogonal injection QqTOF mass spectrometer, making it a suitable technique for phosphoproteomics.     

Absolute quantification of proteins in solutions and in polyacrylamide gels by mass spectrometry

A combination of nanoelectrospray tandem mass spectrometry and (18)O-labeled peptide internal standards was applied for the absolute quantification of proteins from their in-solution and in-gel tryptic digests. Although absolute quantification from in-solution digests was accurate, we observed that in-gel digestion compromised the quantification accuracy by affecting the recovery of individual peptides and, therefore, the provided estimates might be strongly influenced by the selection of reference peptides. Under optimized experimental conditions, it was possible to provide a semiquantitative estimate of the absolute amount of gel separated proteins within better than 50% error margin.     

MALDI analysis of Bacilli in spore mixtures by applying a quadrupole ion trap time-of-flight tandem mass spectrometer

A novel ion trap time-of-flight hybrid mass spectrometer (qIT-TOF MS) has been applied for peptide sequencing in proteolytic digests generated from spore mixtures of Bacilli. The method of on-probe solubilization and in situ proteolytic digestion of small, acid-soluble spore proteins has been recently developed in our laboratory, and microorganism identification in less than 20 min was accomplished. In this study, tryptic peptides were generated in situ from complex spore mixtures of B. subtilis 168, B. globigii, B. thuringiensis subs. Kurstaki, and B. cereus T, respectively. MALDI analysis of bacterial peptides generated was performed with an average mass resolving power of 6200 and a mass accuracy of up to 10 ppm using a trap-TOF tandem configuration. Precursor ions of interest were usually selected and stored in the quadrupole ion trap with their complete isotope distribution by choosing a window of +/- 2 Da. Sequence-specific information on isolated protonated peptides was gained via tandem MS experiments with an average mass resolving power of 4450 for product ion analysis, and protein and bacterial sources were identified by database searching.     

Structural characterization of protein tryptic peptides via liquid chromatography/mass spectrometry and collision-induced dissociation of their doubly charged molecular ions

The formation of multiply charged molecular ions via the field-assisted ion evaporation mechanism during electrospray ionization enables the use of an atmospheric pressure ionization quadrupole mass spectrometer system for characterizing biologically important peptides. The straightforward implementation of high-performance liquid chromatography (HPLC) into this new strategy to determine the molecular weight of tryptic peptides via the pneumatically assisted electrospray (ion spray) interface is presented. Examples utilizing both microbore (1.0 mm) and standard bore (4.6 mm) inside diameter columns are shown for the LC/MS molecular weight determination of tryptic peptides in methionyl-human growth hormone (met-hGH). Injected levels from 50 to 75 pmol of tryptic digest onto 1 mm i.d. HPLC columns provided full-scan LC/MS or LC/MS/MS results without postcolumn splitting of the effluent. When standard 4.6 mm i.d. HPLC columns were used, a 20:1 postcolumn split was utilized, which required from 1 to 5 nmol of injected tryptic digest for full-scan LC/MS or LC/MS/MS results. Collision-induced dissociation (CID) mass spectra resulting from either "infusion" or on-line LC/MS/MS analysis of the abundant doubly charged ions that predominate for tryptic peptides under electrospray conditions provided structurally useful sequence information for met-hGH and human hemoglobin tryptic digests. The slower mass spectrometer scan rate used during infusion of sample provides more accurate mass assignments than on-line LC/MS or LC/MS/MS, but the latter on-line experiments preclude ambiguities caused by matrix or component interferences. However, in some instances very weak CID product ions preclude complete tryptic peptide structural characterization based upon the CID data alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Integrated sample processing system involving on-column protein adsorption, sample washing, and enzyme digestion for protein identification by LC-ESI MS/MS

An automated system has been developed for protein identification using mass spectrometry that incorporates sample cleanup, preconcentration, and protein digestion in a single stage. The procedure involves the adsorption of a protein or a protein mixture from solution onto a hydrophobic medium that is contained within a microcolumn. The protein is digested while still bound to the hydrophobic support. The peptides are then eluted from surface digestion to an electrospray ionization mass spectrometer for detection and sequencing. The entire system is fully automated wherein the mass spectrometer is collecting data continuously. We demonstrate that this system is capable of identifying standard protein samples at concentrations down to 100 nM. Further development of this technique may offer a potential solution for proteomics applications that require unattended operation, such as on-line monitoring and identification of microorganisms on the basis of the detection of their protein biomarkers.     

Integrated microfluidic device for mass spectrometry-based proteomics and its application to biomarker discovery programs

The present investigation describes the analytical performances of a microfluidic device comprising an enrichment column, a reversed-phase separation channel, and a nanoelectrospray emitter embedded altogether in polyimide layers. This configuration minimizes transfer lines and connections and reduces postcolumn peak broadening and dead volumes. This compact and versatile modular nanoLC-chip system was interfaced to both ion trap and time-of-flight mass spectrometers, and its analytical potentials were evaluated in the context of proteomics applications. The figures of merit of this system in terms of peak capacity, reproducibility, sensitivity, and linear dynamic range of peptide detection were determined using tryptic digests of complex protein extracts including albumin- and immunoglobulin-depleted rat plasma samples. The analysis of peak profiles for more than 600 peptide ions reproducibly detected across replicate nanoLC-chip-MS runs (n = 10) indicated that this system provided good reproducibility of retention time and peak intensity with RSD values of less than 0.5 and 9.1%, respectively. Variation in peptide abundance as low as 2-fold changes was identified for spiked tryptic digests present at levels of 2-5 fmol in plasma samples. Sensitivity measurements were performed on dilution series of protein digests spiked into rat plasma samples and provided a detection limit of 1-5 fmol. The modular concept of the microfluidic systems also facilitated the integration of two-dimensional chromatography (strong cation exchange/C18) thereby increasing the sample loading and selectivity of the nanoLC-chip-MS system. The application of this integrated device was evaluated for complex rat plasma samples to compare the number of protein identifications obtained using one- and two-dimensional nanoLC-chip-MS/MS.     

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.     

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.     

Demonstration of direct bioanalysis of drugs in plasma using nanoelectrospray infusion from a silicon chip coupled with tandem mass spectrometry

Quantitative bioanalysis by direct nanoelectrospray infusion coupled to tandem mass spectrometry has been achieved using an automated liquid sampler integrated with an array of microfabricated electrospray nozzles allowing rapid, serial sample introduction (1 min/ sample). Standard curves prepared in human plasma for verapamil (r2 = 0.999) and its metabolite norverapamil (r2 = 0.998) were linear over a range of 2.5-500 ng/ mL. Based on the observed precision and accuracy, a lower limit of quantitation of 5 ng/mL was assigned for both analytes. Sample preparation consisted of protein precipitation with an organic solvent containing the structural analogue gallopamil as an internal standard. Protein precipitation was selected both to maximize throughput and to test the robustness of direct nanoelectrospray infusion. Aliquots of supernatant (10 pL) were transferred to the back plane of the chip using disposable, conductive pipet tips for direct infusion at a flow rate of 300 nL/min. Electrospray ionization occurred from the etched nozzles (30-microm o.d.) on the front of the chip, initiated by a voltage applied to the liquid through the pipet tip. The chip was positioned near the API sampling orifice of a triple quadrupole mass spectrometer, which was operated in selected reaction monitoring mode. Results are presented that document the complete elimination of system carry-over, attributed to lack of a redundant fluid path. This technology offers potential advantages for MS-based screening applications in drug discovery by reducing the time for methods development and sample analysis.     

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.     

A microdevice with integrated liquid junction for facile peptide and protein analysis by capillary electrophoresis/electrospray mass spectrometry

A novel microfabricated device was implemented for facile coupling of capillary electrophoresis with mass spectrometry (CE/MS). The device was constructed from glass wafers using standard photolithographic/wet chemical etching methods. The design integrated (a) sample inlet ports, (b) the separation channel, (c) a liquid junction, and (d) a guiding channel for the insertion of the electrospray capillary, which was enclosed in a miniaturized subatmospheric electrospray chamber of an ion trap MS. The replaceable electrospray capillary was precisely aligned with the exit of the separation channel by a microfabricated guiding channel. No glue was necessary to seal the electrospray capillary. This design allowed simple and fast replacement of either the microdevice or the electrospray capillary. The performance of the device was tested for CE/MS of peptides, proteins, and protein tryptic digests. On-line tandem mass spectrometry was used for the structure identification of the protein digest products. High-efficiency/high-resolution separations could be obtained on a longer channel (11 cm on-chip) microdevice, and fast separations (under 50 s) were achieved with a short (4.5 cm on-chip) separation channel. In the experiments, both electrokinetic and pressure injections were used. The separation efficiency was comparable to that obtained from conventional capillary electrophoresis.     

Interfacing a polymer-based micromachined device to a nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometer

Here we report the design, fabrication, and operation of a polymer-based microchip device interfaced to a nanoelectrospray ionization source and a Fourier transform ion cyclotron resonance mass spectrometer. The poly(methyl methacrylate) micromachined device was fabricated using X-ray lithography to produce a network of channels with high aspect ratios. Fabrication of high aspect ratio channels allows for zero dead volume interfaces between the microchip platform and the nanoelectrospray capillary interface. The performance of this device was evaluated with standard peptide and protein samples. High-quality mass spectral data from peptide and proteins (and mixtures thereof) were obtained without any interfering chemical noise from the polymer or the developers and plasticizers used in the fabrication process. Sample cross-contamination is not a problem using this polymer-based microchip device as demonstrated by the sequential analysis of several proteins. The nanoelectrospray source was operated at flow rates from 20 to 100 nL/min using pressure-driven flow, and uninterrupted operation for several hours is demonstrated without any noticeable signal degradation. The ability to fabricate multiple devices using injection molding or hot-embossing techniques of polymers provides a lower cost alternative to silica-based devices currently utilized with mass spectrometry.     

Design and performance of an ESI interface for selective external ion accumulation coupled to a Fourier transform ion cyclotron mass spectrometer

The coupling of Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) with electrospray ionization has advanced the analysis of large biopolymers and provided the basis for high-throughput protein characterization (e.g., for rapid "proteome" analyses). In this work, the combination of high-performance capillary liquid chromatography with FTICR mass spectrometry and external ion accumulation has been shown to increase both sensitivity and analysis duty cycle. Instrument versatility is further improved by ion preselection followed by ion accumulation in an external linear quadrupole ion trap. The interface was tested with a 3.5-T FTICR mass spectrometer and evaluated with a number of peptides and proteins whose molecular weights ranged from 500 to 66000. A significant increase in the sensitivity, duty cycle, and dynamic range over that of the previously used accumulated trapping was achieved, exhibiting a detection limit of approximately 10 zmol (approximately 6000 molecules) for smaller proteins such as cytochrome c. Capillary LC external accumulation interface with FTICR was successfully applied for the study of whole-proteome mouse tryptic digests.     

Liquid chromatography-atmospheric pressure electron capture dissociation mass spectrometry for the structural analysis of peptides and proteins

Atmospheric pressure electron capture dissociation (AP-ECD) is an emerging technique with the potential to be a more accessible alternative to conventional ECD/electron transfer dissociation (ETD) methods because it can be implemented using a stand-alone ion source device suitable for use with any existing or future electrospray ionization mass spectrometer. With AP-ECD, no modification of the main instrument is required, so it may easily be retrofitted to instruments not originally equipped with ECD/ETD capabilities. Here, we present our first purpose-built AP-ECD source and demonstrate its use in conjunction with capillary LC for the analysis of substance P, a tryptic digest of bovine serum albumin, and a phosphopeptide mixture. Quality ECD spectra were obtained for all the samples at the low femtomole level, proving that LC-AP-ECD-MS is suitable for the structural analysis of peptides and protein digests, in this case using an unmodified quadrupole time-of-flight mass spectrometer built ca. 2002.