Hydrophilic interaction chromatography coupled to electrospray mass spectrometry for the separation of peptides and protein digests

In this study, the analysis of a peptide set, chosen for their differences in hydrophilicity, and the tryptic digests of bovine cytochrome c and β-lactoglobulin by hydrophilic interaction chromatography–electrospray ionisation mass spectrometry (HILIC–ESI-MS) is demonstrated. Two different types of HILIC phases, i.e., an amide- and an amino-modified silica-based phase, packed into narrow bore or capillary columns, were investigated with separations conducted under either low pH or neutral pH conditions. The separation performance of the two HILIC columns with respect to peak efficiency and selectivity have been documented under these different mobile phase conditions, and the results compared with the performance of a conventional capillary reversed-phase C18 column of similar dimensions. It was found that very good separation of the peptide set could be achieved by using the amide-modified silica column over a broad pH range. Moreover, with the protein digest samples, excellent separation of the tryptic digests was obtained with the amide-modified HILIC column under neutral pH conditions. Compared to the conventional reversed-phase C18 separations, the use of these HILIC columns not only provided complementary separation selectivity, but also offered the capability to identify unique peptides using tandem HILIC–mass spectrometric techniques. These studies therefore highlight the potential of HILIC procedures for future proteomic applications.     

Charge state separation for protein applications using a quadrupole time-of-flight mass spectrometer

A novel method for separating ions according to their charge state using a quadrupole time-of-flight mass spectrometer is presented. The benefits of charge state separation are particularly apparent in protein identification applications at low femtomole concentration levels, where in conventional TOF MS spectra peptide ions are often lost in a sea of chemical noise. When doubly and triply charged tryptic peptide ions need to be filtered from singly charged background ions, the latter are suppressed by two to three orders of magnitude, while from 10-50% of multiply charged ions remain. The suppression of chemical noise reduces the need for chromatography and can make this experimental approach the electrospray equivalent of conventional MALDI peptide maps. If unambiguous identification cannot be achieved, MS/MS experiments are performed on the precursor ions identified through charge separation, while the previously described Q2-trapping duty cycle enhancement is tuned for approximately 1.4 of the precursor m/z to enhance intensities of ions with m/z values above that of the precursor. The resulting product ion spectra contain few fragments of impurities and provide quick and unambiguous identification through database search. The multiple charge separation technique requires minimal tuning and may become a useful tool for analysis of complex mixtures.     

Rapid hydrocarbon analysis using a miniature rectilinear ion trap mass spectrometer

A miniature ion trap mass analyzer was applied to the analysis of traces of hydrocarbons and simple heteroatomics in the vapor phase and in aqueous solution. Vapors of acetone, acetic acid, acetonitrile, benzene, butanethiol, carbon disulfide, hexane, dichloromethane, naphthalene, toluene and xylenes were detected and quantified using solid sorbent trapping and, in some cases, by passage through a membrane interface. Aqueous solutions of benzene, toluene, xylenes, hexane and a petroleum naphtha distillate were examined using the membrane interface. Sampling, detection and identification of all compounds was completed in times of less than one minute. The gas-phase samples of toluene and benzene were detected at 200 ppt (limit of detection, LOD) for toluene and 600 ppt for benzene. Identification of benzene and xylene in aqueous solutions was readily achieved with LODs of 200 and 400 ppb, respectively. Quantification over a linear dynamic range of two orders of magnitude for the aqueous samples and three orders of magnitude for the vapor-phase samples was demonstrated.     

Chiral separation of fluorescamine-labeled amino acids using microfabricated capillary electrophoresis devices for extraterrestrial exploration

Chiral separations of fluorescamine-labeled amino acids are characterized and optimized on a microfabricated capillary electrophoresis (CE) device. A standard mixture of acidic and neutral amino acids is labeled with fluorescamine in less than 5 min and the hydroxypropyl-beta-cyclodextrin (HPbetaCD) concentration, temperature, and pH are optimized (15 mM HPbetaCD, 6 degrees C, pH < 9) to achieve high-quality and low background chiral separations in less than 200 s. All four stereoisomers formed in the labeling reaction of the chiral dye with the chiral amino acids are typically resolved. At pH > 9, isomerization of the dye chiral center is observed that occurs on the time scale of the chip separation. Typical limits of detection are approximately 50 nM. These results demonstrate the feasibility of combining fluorescamine labeling of amino acids with microfabricated CE devices to develop low-volume, high-sensitivity apparatus and methods for extraterrestrial exploration.     

Rapid and highly sensitive protein extraction via cobalt oxide nanoparticle-based liquid-liquid microextraction coupled with MALDI mass spectrometry

A new approach for rapid and highly sensitive protein extraction using cobalt oxide nanoparticles modified with cetyltrimethylammonium (Co(3)O(4)/CTA(+) NP) using nanoparticle-based liquid-liquid microextraction (NP-LLME) coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been successfully demonstrated. For the first time, the metal oxide NPs (Co(3)O(4)/CTA(+) NP) prepared in the organic phase (toluene) have been successfully applied for the extraction and preconcentration of proteins from sample solutions and complex samples via electrostatic forces involved between the metal oxide NP and proteins. Lysozyme was used as the model protein to investigate the optimal extraction parameters of the current approach. The optimal conditions were obtained at pH > pI for 10 min of incubation time (extraction time) with 3% salt (NaCl) addition. The Co(3)O(4)/CTA(+) NP was successfully applied for the highly sensitive analysis of an array proteins such as insulin, chymotrypsinogen and lysozyme from aqueous solution, protein mixture and milk samples in nanoparticle-based liquid-phase microextraction coupled with MALDI-MS. The potentiality of the NP-LLME using Co(3)O(4)/CTA(+) NP for the extraction of proteins was also compared with other types of NP-liquid phase microextraction (LPME) methods. The current approach offers distinct advantages including rapidity, straightforwardness, high sensitivity for washing- and separation-free MALDI-MS analysis of proteins.     

Rapid separation and highly sensitive detection methodology for sulfonamides in shrimp using a monolithic column coupled with BDD amperometric detection

In this report, we aimed to extend our previous efforts toward the evaluation of sulfonamides (SAs) with a boron-doped diamond (BDD) electrode. We improved this method by reducing the analysis time using a monolithic column coupled with amperometric detection to determine seven sulfonamides (sulfaguanidine, sulfadiazine, sulfamethazine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline). Because of its rapid separation, low back-pressure and high separation efficiency compared to a particle-packed column, a monolithic column (100 mm × 4.6 mm) was used for sulfonamide separation. Chromatographic separation was performed in less than 8 min. The analysis was carried out using phosphate buffer (0.1 M, pH 3): acetonitrile: methanol in a ratio of 80:15:5 (v/v/v) as the mobile phase with a flow rate of 1.5 mL min⁻¹. The optimal detection potential using hydrodynamic voltammetry was found to be 1.2 V versus Ag/AgCl. The method was applied to determine seven sulfonamides in shrimp after sample preparation by solid-phase extraction. The recoveries of the sulfonamides in spiked shrimp samples at 1.5, 5 and 10 μg g⁻¹ were in the range of 81.7 to 97.5% with a relative standard deviation (R.S.D.) between 1.0 and 4.6%. Our methodology produced results that were highly correlated with HPLC-MS data. Therefore, we propose a method that can be used for the rapid, selective and sensitive evaluation of sulfonamides in contaminated food.     

Rapid protein identification using a microscale electrospray LC/MS system on an ion trap mass spectrometer

A methodology has been developed for the rapid identification of gel separated proteins. Following in gel protein digestion with trypsin, the resulting peptide mixture is analyzed by on-line liquid chromatography, electrospray mass spectrometry (LC/MS). The mass spectral data containing either accurate mass values or sequence specific fragment ion information is then matched to a database of known protein sequences. Key features of the LC/MS system are the use of a novel integrated, microscale LC column-electrospray interface and variable flow solvent delivery to optimize the efficiency of sample loading and gradient elution. With these enhancements, only 10 min is required to analyze each sample. The method is routine for sample amounts ranging from 50 to 500 fmol. The analysis parameters for the ion trap mass spectrometer have to be carefully adjusted in order to keep pace with the rapidly eluting LC peaks. Although designed for rapid LC separations, the integrated column-electrospray interface is also able to provide extended analyses of selected components using a technique known as “peak parking. ”     

Capabilities of high resolution inductively coupled plasma mass spectrometry for trace element determination in plant sample digests

The analytical performance of inductively coupled plasma mass spectrometry (ICP-MS) for the analysis of plant sample digests was evaluated using double focusing sector field ICP-MS (ICP-SMS). Instrumental detection limits of ICP-SMS are superior to those obtained by quadrupole systems (ICP-QMS) and reach the fg mL^–1 range for elements with high m/z ratios. Matrix effects caused by a plant digest after sample preparation resulting in 200-fold dilution were found to be negligible. The usefulness of high mass resolution for overcoming some spectral interferences is demonstrated. Mathematical correction possibilities could be necessary to improve accuracy. The concentrations of more than 20 elements can be determined in 5 min and only one internal standard is necessary to correct for instrumental drift. Received: 30 March 1998 / Revised: 4 May 1998 / Accepted: 16 May 1998     

Capabilities of high resolution inductively coupled plasma mass spectrometry for trace element determination in plant sample digests

The analytical performance of inductively coupled plasma mass spectrometry (ICP-MS) for the analysis of plant sample digests was evaluated using double focusing sector field ICP-MS (ICP-SMS). Instrumental detection limits of ICP-SMS are superior to those obtained by quadrupole systems (ICP-QMS) and reach the fg mL^–1 range for elements with high m/z ratios. Matrix effects caused by a plant digest after sample preparation resulting in 200-fold dilution were found to be negligible. The usefulness of high mass resolution for overcoming some spectral interferences is demonstrated. Mathematical correction possibilities could be necessary to improve accuracy. The concentrations of more than 20 elements can be determined in 5 min and only one internal standard is necessary to correct for instrumental drift. Received: 30 March 1998 / Revised: 4 May 1998 / Accepted: 16 May 1998     

Uranium isotopic analyses by using a sector field inductively coupled plasma mass spectrometer

A sector field mass spectrometer using an inductively coupled plasma as an ion source was used in order to determine the uranium isotopic ratios in reference materials supplied by the National Institute of Standards and Technology (NIST) (NBS 950, 010, 030, 200, 500, 750, and 970). The accuracy obtained for the major isotopes was better than 0.2%.     

Analysis of plasma protein adsorption onto DC-Chol-DOPE cationic liposomes by HPLC-CHIP coupled to a Q-TOF mass spectrometer

Plasma protein adsorption is regarded as a key factor in the in vivo organ distribution of intravenously administered drug carriers, and strongly depends on vector surface characteristics. The present study aimed to characterize the “protein corona” absorbed onto DC-Chol-DOPE cationic liposomes. This system was chosen because it is one of the most efficient and widely used non-viral formulations in vitro and a potential candidate for in vivo transfection of genetic material. After incubation of human plasma with cationic liposomes, nanoparticle–protein complex was separated from plasma by centrifugation. An integrated approach based on protein separation by one-dimensional 12% polyacrylamide gel electrophoresis followed by the automated HPLC-Chip technology coupled to a high-resolution mass spectrometer was employed for protein corona characterization. Thirty gel lanes, approximately 2 mm, were cut, digested and analyzed by HPLC-MS/MS. Fifty-eight human plasma proteins adsorbed onto DC-Chol-DOPE cationic liposomes were identified. The knowledge of the interactions of proteins with liposomes can be exploited for future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins in body fluids.

An enhanced microfluidic chip coupled to an electrospray Qstar mass spectrometer for protein identification

The combination of microfabricated fluidic systems (muFAB) and electrospray mass spectrometers (ESI-MS) will provide multiplexed and integrated analytical systems for proteins and other biomolecules. Implementation of this novel approach requires the development of robust and user-friendly muFAB devices. Here, we present new approaches that improve the robustness, user friendliness and performance of muFAB devices coupled to MS. First, we present the development of a convenient mount to connect a muFAB device to the ESI-MS and the incorporation of filters in the reservoirs and exit of the muFAB. This mount facilitates interfacing and significantly reduces the chemical noise observed by the MS. Furthermore, we demonstrate improvements in sample handling and delivery by using either a nonaqueous electrolyte or a cationic coating on the surfaces in the muFAB device and transfer capillary. These improvements are applied to protein analysis by continuous infusion of proteolytic digests.     

Rapid screening and characterization of drug metabolites using a new quadrupole-linear ion trap mass spectrometer

The application of a new hybrid RF/DC quadrupole-linear ion trap mass spectrometer to support drug metabolism and pharmacokinetic studies is described. The instrument is based on a quadrupole ion path and is capable of conventional tandem mass spectrometry (MS/MS) as well as several high-sensitivity ion trap MS scans using the final quadrupole as a linear ion trap. Several pharmaceutical compounds, including trocade, remikiren and tolcapone, were used to evaluate the capabilities of the system with positive and negative turbo ionspray, using either information-dependent data acquisition (IDA) or targeted analysis for the screening, identification and quantification of metabolites. Owing to the MS/MS in-space configuration, quadrupole-like CID spectra with ion trap sensitivity can be obtained without the classical low mass cutoff of 3D ion traps. The system also has MS(3) capability which allows fragmentation cascades to be followed. The combination of constant neutral loss or precursor ion scan with the enhanced product ion scan was found to be very selective for identifying metabolites at the picogram level in very complex matrices. Owing to the very high cycle time and, depending on the mass range, up to eight different MS experiments could be performed simultaneously without compromising chromatographic performance. Targeted product ion analysis was found to be complementary to IDA, in particular for very low concentrations. Comparable sensitivity was found in enhanced product ion scan and selected reaction monitoring modes. The instrument is particularly suitable for both qualitative and quantitative analysis.     

Rapid and quantitative extraction and analysis of trace organics using directly coupled SFE-GC

Supercritical fluid extraction can be coupled with capillary gas chromatography (SFE-GC) using commercially-available on-column or split/splitless injection ports. While liquid solvent extractions require several hours or even days to perform, SFC-GC analyses can be completed in ≤ 1 hour including extraction, analyte concentration, and GC separation. SFE-GC yields chromatographic peak shapes that compare favorably with those obtained using conventional liquid solvent injections. Quantitative extraction and recovery of analytes is usually achieved in 10 minutes, and maximum sensitivity is obtained since the extracted analytes can be quantitatively transferred into the GC column for cryogenic focusing prior to GC analysis. SFE-GC analysis of a variety of organic pollutants from environmental solids and sorbent resins, and flavor and fragrance compounds from food products will be discussed.     

Rapid protein digestion and identification using monolithic enzymatic microreactor coupled with nano-liquid chromatography-electrospray ionization mass spectrometry

A novel monolithic enzymatic microreactor was prepared in the fused-silica capillary by in situ polymerization of acrylamide (AA), N-acryloxysuccinimide (NAS) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, which could offer very low back pressure, enabling the fast digestion of proteins. The performance of the monolithic microreactor was demonstrated by digesting cytochrome c at high flow rate, and the comparisons between the in-solution digestion and on-column reaction were made by a nano-high performance liquid chromatography-mass spectrometry (nano-HPLC-MS) system. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at the fast flow rate of 1 microL/min, which afforded a residence time of 7s, yielding a sequence coverage of 54.81% using strict multiple database searching thresholds. Future more, a mixture of four standard proteins was digested and analyzed using the on-line digestion and nano-HPLC-MS system. The results showed the promising of such a system in the analysis of protein mixture.     

Isotope measurements in uranium using a quadrupole inductively coupled plasma mass spectrometer (ICPMS)

A series of measurements were carried out to establish the reliability associated with isotope ratio (235/238) measurements on uranium samples using a quadrupole inductively coupled plasma mass spectrometer (ICPMS). Figures of merit related to the isotopic measurements were determined using non certified as well as certified materials provided by the New Brunswick Laboratory (NBL). The experimental results showed that repeatability is around 0.5% while reproducibility was calculated as 0.27%. Mass discrimination was determined as 0.03% per mass unit and the system linearity check over five orders of isotope ratios yielded a mass discrimination factor (K factor) of 1.0002±0.0081 (0.81%, 2s). The mean error of measurement obtained from six different certified reference materials was 0.77%.     

Chip-based CE coupled to a quadrupole TOF mass spectrometer for the analysis of a glycopeptide

A novel approach using sheath flow chip-based CE coupled to a quadrupole TOF mass spectrometer was developed and applied to the analysis of an O-glycopeptide. The method provided good separation and sensitive analysis of the glycopeptide and its beta-elimination product. The beta-elimination reaction of the glycopeptide with ammonia or dimethylamine was adapted for changing the glycopeptide into deglycosylated form and for specifying the site of glycosylation. MS/MS analysis of the native peptide, glycopeptide and beta-elimination product enabled the identification of the glycosylation site through the comparison of the fragmentations and peptide sequence analysis.