De novo sequencing of unique sequence tags for discovery of post-translational modifications of proteins

De novo sequencing is a spectrum analysis approach for mass spectrometry data to discover post-translational modifications in proteins; however, such an approach is still in its infancy and is still not widely applied to proteomic practices due to its limited reliability. In this work, we describe a de novo sequencing approach for the discovery of protein modifications based on identification of the proteome UStags (Shen, Y.; Toli?, N.; Hixson, K. K.; Purvine, S. O.; Pasa-Toli?, L.; Qian, W. J.; Adkins, J. N.; Moore, R. J.; Smith, R. D. Anal. Chem. 2008, 80, 1871-1882). The de novo information was obtained from Fourier-transform tandem mass spectrometry data for peptides and polypeptides from a yeast lysate, and the de novo sequences obtained were selected based on filter levels designed to provide a limited yet high quality subset of UStags. The DNA-predicted database protein sequences were then compared to the UStags, and the differences observed across or in the UStags (i.e., the UStags' prefix and suffix sequences and the UStags themselves) were used to infer possible sequence modifications. With this de novo-UStag approach, we uncovered some unexpected variances within several yeast protein sequences due to amino acid mutations and/or multiple modifications to the predicted protein sequences. To determine false discovery rates, two random (false) databases were independently used for sequence matching, and ~3% false discovery rates were estimated for the de novo-UStag approach. The factors affecting the reliability (e.g., existence of de novo sequencing noise residues and redundant sequences) and the sensitivity of the approach were investigated and described. The combined de novo-UStag approach complements the UStag method previously reported by enabling the discovery of new protein modifications.     

Technical updates to the bacterial method for nitrate isotopic analyses

The bacterial conversion of aqueous nitrate (NO(3)(-)) to nitrous oxide (N(2)O) for isotopic analysis has found widespread use since its introduction (Sigman, D. M.; Casciotti, K. L.; Andreani, M.; Galanter, M.; Bo?hlke, J. K. Anal. Chem.2001, 73, 4145-4153; Casciotti, K. L.; Sigman, D. M.; Galanter Hastings, M.; Bo?hlke, J. K.; Hilkert, A. Anal. Chem.2002, 74, 4905-4912). The bacterial strain Pseudomonas aureofaciens (ATTC no. 13985) was shown to convert NO(3)(-) to N(2)O while retaining both N and O isotopic signatures, and automation of the isotopic analysis of N(2)O greatly increased the throughput of the method (Casciotti, K. L.; Sigman, D. M.; Galanter Hastings, M.; Bo?hlke, J. K.; Hilkert, A. Anal. Chem.2002, 74, 4905-4912). Continued development of the denitrifier method has led to increased precision and throughput of NO(3)(-) isotopic analysis. Presented here are several recent procedural modifications and the demonstration of their effectiveness.     

Ultrasensitive proteomics using high-efficiency on-line micro-SPE-nanoLC-nanoESI MS and MS/MS

Ultrasensitive nanoscale proteomics approaches for characterizing proteins from complex proteomic samples of <50 ng of total mass are described. Protein identifications from 0.5 pg of whole proteome extracts were enabled by ultrahigh sensitivity (<75 zmol for individual proteins) achieved using high-efficiency (peak capacities of approximately 10(3)) 15-microm-i.d. capillary liquid chromatography separations (i.e., using nanoLC, approximately 20 nL/min mobile-phase flow rate at the optimal linear velocity of approximately 0.2 cm/s) coupled on-line with a micro-solid-phase sample extraction and a nanoscale electrospray ionization interface to a 11.4-T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS). Proteome measurement coverage improved as sample size was increased from as little as 0.5 pg of sample. It was found that a 2.5-ng sample provided 14% coverage of all annotated open reading frames for the microorganism Deinococcus radiodurans, consistent with previous results for a specific culture condition. The estimated detection dynamic range for detected proteins was 10(5)-10(6). An improved accurate mass and LC elution time two-dimensional data analysis methodology, used to both speed and increase the confidence of peptide/protein identifications, enabled identification of 872 proteins/run from a single 3-h nanoLC/FTICR MS analysis. The low-zeptomole-level sensitivity provides a basis for extending proteomics studies to smaller cell populations and potentially to a single mammalian cell. Application with ion trap MS/MS instrumentation allowed protein identification from 50 pg (total mass) of proteomic samples (i.e., approximately 100 times larger than FTICR MS), corresponding to a sensitivity of approximately 7 amol for individual proteins. Compared with single-stage FTICR measurements, ion trap MS/MS provided a much lower proteome measurement coverage and dynamic range for a given analysis time and sample quantity.     

Detection of multiphosphorylated peptides in LC-MS/MS analysis under low pH conditions

An improved method of detection of multiphosphorylated peptides by RPLC-MS/MS analysis under low pH conditions (pH approximately 1.7, 3% formic acid) is demonstrated for the model phosphoproteins, bovine alpha- and beta-casein. Changes in the pH conditions from normal (pH approximately 3.0, 0.1% formic acid) to low (pH approximately 1.7, 3% formic acid) significantly improved the detection limit of multiphosphorylated peptides carrying negative (-) solution charge states. In particular, bovine beta-casein tetraphosphorylated peptide, was detected with a loading amount of only 50 fmol of trypsin-digested bovine beta-casein under low pH conditions, which is 200 times lower than necessary to detect the peptide under normal pH conditions. In order to understand the low pH effect, various loading amounts of trypsin-digested bovine alpha- and beta-caseins were analyzed by RPLC-MS/MS analyses under two different pH conditions. The question of whether the low pH condition improves the detection of multiphosphorylated peptides by increasing ionization efficiencies could not be proven in this study because synthetic multiphosphorylated peptides could not be easily obtained by peptide synthesis. Interestingly, increased hydrophilicity resulting from multiple phosphorylation events is shown to negatively affect the peptide retention on reversed-phase column material. It was also demonstrated that the low pH condition could effectively enhance the retention of multiphosphorylated peptides on reversed-phase column material. The usefulness of low pH RPLC analysis was tested using an actual phosphopeptide-enriched sample prepared from mouse brain tissues. Previously, low pH solvents have been used in SCX fractionation and TiO2 enrichment processes to selectively enrich phosphopeptides during the phosphopeptide enrichment procedure, but the improved detection of multiphosphorylated peptides in RPLC-MS/MS analysis under low pH conditions has not been reported before (Ballif, B. A.; Villen, J.; Beausoleil, S. A.; Schwartz, D.; Gygi, S. P. Mol. Cell. Proteomics 2004, 3, 1093-1101. Villen, J.; Beausoleil, S. A.; Gerber, S. A.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 1488-1493. Schlosser, A.; Vanselow, J. T.; Kramer, A. Anal. Chem. 2005, 77, 5243-5250.).     

An automated multidimensional protein identification technology for shotgun proteomics.

We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column. We detail the improvements over a system described by Link et al. (Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682) that separates and acquires tandem mass spectra for thousands of peptides. Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis. In addition, we describe the chromatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% between two analyses. Furthermore, a dynamic range of 10000 to 1 between the most abundant and least abundant proteins/peptides in a complex peptide mixture has been demonstrated. By improving sample preparation along with separations, the method improves the overall analysis of proteomes by identifying proteins of all functional and physical classes.     

Structural characterization of chemically derivatized oligosaccharides by nanoflow electrospray ionization mass spectrometry.

Oligosaccharides released from several glycoproteins were derivatized with either 4-aminobenzoic acid 2-(diethylamino)ethyl ester (ABDEAE) (Yoshino, K.; et al. Anal. Chem. 1995, 67, 4028-4031) or 2-aminopyridine. The resulting derivatives were analyzed on a nanoflow electrospray ionization (ESI) quadrupole-inlet time-of-flight mass spectrometer using the low-energy collision-induced dissociation technique. In the MS/MS spectra, the oxonium (b or internal series) and y series ions, which are derived from the multiply charged precursor ions, were predominant and were used for the structural readout. Some oxonium ions that were observed in the low-mass region, but that were not found in the PSD analyses (Mo, W.; et al. Anal. Chem. 1998, 70, 4520-4526), rendered a more detailed structural insight. The oxonium ions at m/z 512.2, which are derived from the fucosylated oligosaccharides of immunoglobulin Y and thyroglobulin, were observed, suggesting that fucosylation had occurred proximal to the outer nonreducing terminus. In addition, the data herein show that structural elucidation can be routinely achieved at a low sample concentration. For the case of ABDEAE derivatives, this can be achieved at the 50 fmol/microL level and with the actual sample consumption at the attomole level using nanoflow ESI MS/MS.     

Biomarker profiling and reproducibility study of MALDI-MS measurements of Escherichia coli by analysis of variance-principal component analysis

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has proved useful for the characterization of bacteria and the detection of biomarkers. Key challenges for MALDI-MS measurements of bacteria are overcoming the relatively large variability in peak intensities. A soft tool, combining analysis of variance and principal component analysis (ANOVA-PCA) (Harrington, P. D.; Vieira, N. E.; Chen, P.; Espinoza, J.; Nien, J. K.; Romero, R.; Yergey, A. L. Chemom. Intell. Lab. Syst. 2006, 82, 283-293. Harrington, P. D.; Vieira, N. E.; Espinoza, J.; Nien, J. K.; Romero, R.; Yergey, A. L. Anal. Chim. Acta. 2005, 544, 118-127) was applied to investigate the effects of the experimental factors associated with MALDI-MS studies of microorganisms. The variance of the measurements was partitioned with ANOVA and the variance of target factors combined with the residual error was subjected to PCA to provide an easy to understand statistical test. The statistical significance of these factors can be visualized with 95% Hotelling T2 confidence intervals. ANOVA-PCA is useful to facilitate the detection of biomarkers in that it can remove the variance corresponding to other experimental factors from the measurements that might be mistaken for a biomarker. Four strains of Escherichia coli at four different growth ages were used for the study of reproducibility of MALDI-MS measurements. ANOVA-PCA was used to disclose potential biomarker proteins associated with different growth stages.     

Complete quantitative online analysis of methanol electrooxidation products via electron impact and electrospray ionization mass spectrometry

We report on a novel approach for complete quantitative online product analysis in electrocatalytic reactions, combining electron impact ionization mass spectrometry (EI-MS) and electrospray ionization mass spectrometry (ESI-MS) for simultaneous detection of both volatile and nonvolatile reaction products. The potential of this method is demonstrated using continuous methanol oxidation in a flow cell. The overall reaction rate was followed via the Faradaic current; CO(2) formation was monitored mass spectrometrically via a membrane inlet system, and formaldehyde and formic acid were detected by ESI-MS after a derivatization-extraction-separation procedure introduced recently (Zhao, W.; Jusys, Z.; Behm, R. J. Anal. Chem.2010, 82, 2472-2479) providing quantitative data on the product distribution. In a more general sense, this approach is applicable for a wide range of reactions at the solid-liquid interface or in liquid phase.     

Automated measurement of nitrogen trifluoride in ambient air

We present an analytical method for the in situ measurement of atmospheric nitrogen trifluoride (NF(3)), an anthropogenic gas with a 100-year global warming potential of over 16,000. This potent greenhouse gas has a rising atmospheric abundance due to its emission from a growing number of manufacturing processes and an expanding end-use market. Here we present a modified version of the "Medusa" preconcentration gas chromatography/mass spectrometry (GC/MS) system of Miller, B. R.; Weiss, R. F.; Salameh, P. K.; Tanhua, T.; Greally, B. R.; Mühle, J.; Simmonds, P. G. Anal. Chem.2008, 80 (5), 1536-1545. By altering the techniques of gas separation and chromatography after initial preconcentration, we are now able to make atmospheric measurements of NF(3) with relative precision <2% (1σ) for current background clean air samples. Importantly, this method augments the currently operational Medusa system, so that the quality of data for species already being measured is not compromised and NF(3) is measured from the same preconcentrated sample. We present the first in situ measurements of NF(3) from La Jolla, California made 11 times daily, illustrating how global deployment of this technique within the AGAGE (Advanced Global Atmospheric Gases Experiment) network could facilitate estimation of global and regional NF(3) emissions over the coming years.     

Differential proteomics via probabilistic peptide identification scores

Relative quantitation is key to enable differential proteomics and hence answer biological questions by comparing samples. Classical approaches involve stable isotope labeling with/without spiked standards. Although stable isotopes may lead to precise results, their application is not straightforward. In Proteomics, 2004, 4, 2333-2351, we proposed an approach where we summed peptide identification scores to derive a semiquantitative abundance indicator. In this study, we combine such an indicator with a statistical test to detect differentially expressed proteins. We demonstrate the effectiveness of this method by using mixtures of purified proteins and human plasma spiked with proteins at low-nanomolar concentrations. The impact of the number of repeated experiments is discussed, and we show that the statistical test we use performs well with two to three repetitions, whereas a classical t-test would require at least four repetitions to achieve the same performance. Typically, 2.5-5-fold changes are detected with 90-95% confidence in human plasma. The method is finally characterized by deriving estimates of its false positive and negative rates. This new characterization is valid for a wider class of methods such as spectrum sampling (Liu, H.; Sadygov, R. G.; Yates, J. R. III. Anal. Chem. 2004, 76, 4193-4201).     

Packed column supercritical fluid chromatography/mass spectrometry for high-throughput analysis. Part 2

A supercritical fluid chromatograph was previously interfaced to a mass spectrometer (SFC/MS) and the system evaluated for applications requiring high sample throughput using negative-mode atmospheric-pressure chemical ionization (APCI) (Ventura et al. Anal. Chem. 1999, 71, 2410-2416). This report extends the previous work demonstrating the effectiveness of SFC/MS, using positive ion APCI for the analysis of compounds with a wide range of polarities. Substituting SFC/MS for LC/MS results in substantial time saving, increased chromatographic efficiency, and more precise quantitation of sample mixtures. Flow injection analysis (FIA) also benefits from our SFC/MS system. A broader range of solvents is compatible with the SFC mobile phase compared with LC/MS, and solutes elute more rapidly from the SFC/MS system, reducing sample carryover and cycle time. Our instrumental setup also allows for facile conversion between LC/MS and SFC/MS modes of operation.     

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

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

Development of high-sensitivity ion trap ion mobility spectrometry time-of-flight techniques: a high-throughput nano-LC-IMS-TOF separation of peptides arising from a Drosophila protein extract

A linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome.     

Effect of analyte adsorption on the electroosmotic flow in microfluidic channels

The predictability and constancy over time of the electroosmotic flow in microchannels is an important consideration in microfluidic devices. A common cause for alteration of the flow is the adsorption of analytes to channel walls, for example, during capillary electrophoresis of proteins. It is shown that certain experimental data, published by Towns and Regnier (Towns, J. K; Regnier, F. E. Anal. Chem. 1992, 64, 2473-2478.), on the anomalous elution times for proteins in capillary electrophoresis can be explained using a simple model for analyte adsorption that uses a result first reported by Anderson and Idol (Anderson, J. L.; Idol, W. K Chem. Eng. Commun. 1985, 38, 93-106.) on the electroosmotic flux in capillaries with axial variations in zeta-potential. It is suggested that it might be possible to use such a model to dynamically correct for altered elution times in capillary electrophoretic devices.     

Thermally induced N-to-O rearrangement of tert-N-oxides in atmospheric pressure chemical ionization and atmospheric pressure photoionization mass spectrometry: differentiation of N-oxidation from hydroxylation and potential determination of N-oxidation site

N-Oxides are known to undergo deoxygenation during atmospheric pressure chemical ionization (Ramanathan, R.; Su, A.-D.; Alvarez, N.; Blumenkrantz, N.; Chowdhury, S. K.; Alton, K.; Patrick, J. Anal. Chem. 2000, 72, 1352-1359) resulting from thermal energy activation at the vaporizer of the APCI source. In addition to deoxygenation, tert-N-oxides containing an alkyl or benzyl group on the N-oxide nitrogen also undergo an N-R to O-R rearrangement (Meisenheimer arrangement, where R = alkyl or benzyl), followed by elimination of an aldehyde (or a ketone) through an internal hydrogen transfer. This has been observed under both atmospheric pressure chemical ionization and atmospheric pressure photoionization conditions. These fragment ions were not observed in the product ion spectra from the protonated molecules of the corresponding N-oxides. The elimination of an aldehyde or a ketone, thus, results from thermal energy activation at the vaporizer and is not induced by collisional activation. These fragmentations not only distinguish N-oxides from isomeric hydroxylated metabolites but also provide a potential way to determine the position of N-oxidation when a metabolite (or molecule) contains multiple N-oxidation sites that are in different chemical environments.     

Matrix-assisted laser desorption/ionization fourier transform mass spectrometry for the identification of orcokinin neuropeptides in crustaceans using metastable decay and sustained off-resonance irradiation

Vacuum UV matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance mass spectrometry (FTMS) has been applied to the direct analysis of crustacean neuronal tissues using in-cell accumulation techniques to improve sensitivity. In an extension of previous work by Li and co-workers (Kutz, K. K.; Schmidt, J. J.; Li, L. Anal. Chem. 2004, 76, 5630-5640), and with a focus on the Maine lobster, Homarus americanus, we report that many peaks appearing in direct tissue spectra from crustaceans result from the metastable decay of aspartate-containing neuropeptides with localized protonation sites. We report on mass spectral characteristics of crustacean neuropeptides under MALDI-FTMS conditions and show how fragments formed by Asp-Xxx cleavages can be used to advantage for the identification of orcokinin peptides, a ubiquitous family of crustacean neuropeptides with a highly conserved N-terminus sequence. We show that predicted fragment ion fingerprints (FIFs) can be used to screen internally calibrated direct tissue spectra to provide high-confidence identification of previously identified orcokinin peptides. We use FIFs, identified based upon characteristic neutral losses, to screen for new members of the orcokinin family. Sustained off-resonance irradiation of y-series fragment ions is used to sequence the variable C-terminus. We apply these techniques to the analysis of CoG tissues from Cancer borealis and Panulirus interruptus and show that orcokinins in P. interruptus were misidentified in a previous MALDI-TOF study.     

Hybridization behavior of mixed DNA/alkylthiol monolayers on gold: characterization by surface plasmon resonance and 32P radiometric assay

Nucleic acid assay from a complex biological milieu is attractive but currently difficult and far from routine. In this study, DNA hybridization from serum dilutions into mixed DNA/mercaptoundecanol (MCU) adlayers on gold was monitored by surface plasmon resonance (SPR). Immobilized DNA probe and hybridized target densities on these surfaces were quantified using 32P-radiometric assays as a function of MCU diluent exposure. SPR surface capture results correlated with radiometric analysis for hybridization performance, demonstrating a maximum DNA hybridization on DNA/MCU mixed adlayers. The maximum target surface capture produced by MCU addition to the DNA probe layer correlates with structural and conformational data on identical mixed DNA/MCU adlayers on gold derived from XPS, NEXAFS, and fluorescence intensity measurements reported in a related study (Lee, C.-Y.; Gong, P.; Harbers, G. M.; Grainger, D. W.; Castner, D. G.; Gamble, L. J. Anal. Chem. 2006, 78, 3316-3325.). MCU addition into the DNA adlayer on gold also improved surface resistance to both nonspecific DNA and serum protein adsorption. Target DNA hybridization from serum dilutions was monitored with SPR on the optimally mixed DNA/MCU adlayers. Both hybridization kinetics and efficiency were strongly affected by nonspecific protein adsorption from a complex milieu even at a minimal serum concentration (e.g., 1%). No target hybridization was detected in SPR assays from serum concentrations above 30%, indicating nonspecific protein adsorption interference of DNA capture and hybridization from complex milieu. Removal of nonsignal proteins from nucleic acid targets prior to assay represents a significant issue for direct sample-to-assay nucleic acid diagnostics from food, blood, tissue, PCR mixtures, and many other biologically complex sample formats.     

Broadband coupling into a single-mode,electroactive integrated optical waveguide for spectroelectrochemical analysis of surface-confined redox couples

A single-mode, electroactive waveguiding platform capable of measuring spectroelectrochemical responses of surface-adsorbed redox-active molecules over a broad spectral bandwidth has been created. This new planar waveguide spectrometer is a combination of the previously developed electroactive integrated optical waveguide (EA-IOW; Dunphy, D. R.; Mendes, S. B.; Saavedra, S. S.; Armstrong, N. R. Anal. Chem. 1997, 69, 3086-3094) with a recently reported simplified approach to broadband coupling (Bradshaw, J. T.; Mendes, S. B.; Saavedra, S. S. Anal. Chem. 2002, 74, 1751-1759). With the use of a commercially available prism as an incoupling element, the EA-IOW can now guide visible light from at least 500 to 700 nm, improving upon its previously demonstrated monochromatic nature. Coupling profiles of various laser lines along with transmission spectra of narrow band-pass filters at various potentials are used to demonstrate the optical characteristics of this broadband EA-IOW and to compare its response to that of a conventional transmission instrument. Assessment of spectral resolution, performed by measuring the fwhm of various laser lines, ranges from 0.6 to 0.8. To demonstrate the capabilities of this technology, we show the acquisition of absorbance spectra of two different adsorbates, cytochrome c and ferrocenedicarboxylic acid, as a function of applied potential. Subtleties in the redox chemistries of adsorbed molecules, which were difficult to monitor with a monochromatic waveguide, are readily apparent when using the broadband coupling scheme.