Analysis of quantitative proteomic data generated via multidimensional protein identification technology

We describe the analysis of quantitative proteomic samples via multidimensional protein identification technology (MudPIT). Ratio amounts of the soluble portion of the S. cerevisiae proteome from cultures of S. cerevisiae strain S288C grown in either 14N minimal media or 15N-enriched minimal media were mixed and digested into a complex peptide mixture. A 1 x 14N/1 x 15N complex peptide mixture was analyzed by single-dimensional reversed-phase chromatography and electrospray ionization quadrapole time-of-flight mass spectrometry in order to demonstrate the replacement of 14N by 15N under the growth conditions used. After conformation of the incorporation of 15N into the labeled sample, three separate samples consisting of a 1 x 14N/1 x 15N complex peptide mixture, a 5 x 14N/1 x 15N complex peptide mixture, and a 10 x 14N/1 x 15N complex peptide mixture were analyzed via MudPIT. We demonstrate the dynamic range of the system by analyzing a 1:1, 5:1, and 10:1 data set using the soluble portion from S. cerevisiae grown in either 14N or 15N-enriched minimal media. The method described provides an accurate way to undertake a large-scale quantitative proteomic study.     

Automated ultra-high-pressure multidimensional protein identification technology (UHP-MudPIT) for improved peptide identification of proteomic samples

Multidimensional separation is one of the most successful approaches for proteomics studies that deal with complex samples. We have developed an automated ultra-high-pressure multidimensional liquid chromatography system that operates up to approximately 20 kpsi to improve separations and increase protein coverage from limited amount of samples. The reversed-phase gradient is operated in the constant-flow mode opposed to the constant-pressure mode, which is typical of previous ultra-high-pressure systems. In contrast to constant-pressure systems, the gradient shape is fully controllable and can be optimized for the type of samples to be run. The system also features fast sample loading/desalting using a vented column approach to improve sample throughput. This approach was validated on a soluble fraction from yeast lysate where we achieved approximately 30% more protein identifications using a 60-cm-long triphasic capillary column than with our traditional approach. Advantages of the use of a relatively long reversed-phase column (approximately 50 cm) for MudPIT-type experiments are also discussed.     

Mass measurement accuracy in analyses of highly complex mixtures based upon multidimensional recalibration

Mass spectrometry combined with a range of on-line separation techniques has become a powerful tool for characterization of complex mixtures, including protein digests in proteomics studies. Accurate mass measurements can be compromised due to variations that occur in the course of an on-line separation, e.g., due to excessive space charge in an ion trap, temperature changes, or other sources of instrument "drift". We have developed a multidimensional recalibration approach that utilizes existing information on the likely mixture composition, taking into account variable conditions of mass measurements, and that corrects the mass calibration for sets of individual peaks binned by, for example, the total ion count for the mass spectrum, the individual peak abundance, m/z value, and liquid chromatography separation time. The multidimensional recalibration approach uses a statistical matching of measured masses in such measurements, often exceeding 105, to a significant number of putative known species likely to be present in the mixture (i.e., having known accurate masses), to identify a subset of the detected species that serve as effective calibrants. The recalibration procedure involves optimization of the mass accuracy distribution (histogram), to provide a more confident distinction between true and false identifications. We report the mass accuracy improvement obtained for data acquired using a TOF and several FTICR mass spectrometers. We show that the multidimensional recalibration better compensates for systematic mass measurement errors and also significantly reduces the mass error spread: i.e., both the accuracy and precision of mass measurements are improved. The mass measurement improvement is found to be virtually independent of the initial instrument calibration, allowing, for example, less frequent calibration. We show that this recalibration can provide sub-ppm mass measurement accuracy for measurements of a complex fungal proteome tryptic digest and provide improved confidence or numbers of peptide identifications.     

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.     

RCADiA: simple automation platform for comparative multidimensional protein identification technology

Multidimensional liquid chromatography in combination with tandem mass spectrometry has been used to analyze a variety of biological structures including protein complexes. Incorporating this approach with autosampling devices presents a number of problems including decreased sensitivity due to exposure to extra surfaces, carryover from run to run, and increased dead volume. We developed a device, termed Radial Column Array for Distribution and Automation (RCADiA), to automate multiple MuDPIT experiments while eliminating many of these problems and maintaining a high resolution and sensitive analysis. The design, which places each sample downstream of any common fluid path, presents a low risk of carryover between successive analyses. Beyond the convenience of automation, the RCADiA platform also produces data of similar quality to the standard method of performing individual MuDPIT experiments. We demonstrate this device by performing a comparative analysis of mitochondria enriched from rat liver and spinal cord.     

Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology

A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.     

Acid-labile isotope-coded extractants: a class of reagents for quantitative mass spectrometric analysis of complex protein mixtures

Quantitative mass spectrometry using stable isotope-labeled tagging reagents such as isotope-coded affinity tags has emerged as a powerful tool for identification and relative quantitation of proteins in current proteomic studies. Here we describe an integrated approach using both automated two-dimensional liquid chromatography/ mass spectrometry (2D-LC/MS) and a novel class of chemically modified resins, termed acid-labile isotope-coded extractants (ALICE), for quantitative mass spectrometric analysis of protein mixtures. ALICE contains a thiol-reactive group that is used to capture all cysteine (Cys)-containing peptides from peptide mixtures, an acid-labile linker, and a nonbiological polymer. The acid-labile linker is synthesized in both heavy and light isotope-coded forms and therefore enables the direct relative quantitation of peptides/proteins through mass spectrometric analysis. To test the ALICE method for quantitative protein analysis, two model protein mixtures were fully reduced, alkylated, and digested in solution separately and then Cys-containing peptides covalently captured by either light or heavy ALICE. The reacted light and heavy ALICE were mixed and washed extensively under rigorous conditions and the Cys-containing peptides retrieved by mild acid-catalyzed elution. Finally, the eluted peptides were directly subjected to automated 2D-LC/MS for protein identification and LC/MS for accurate relative quantitation. Our initial study showed that quantitation of protein mixtures using ALICE was accurate. In addition, isolation of Cys-containing peptides by the ALICE method was robust and specific and thus yielded very low background in mass spectrometric studies. Overall, the use of ALICE provides improved dynamic range and sensitivity for quantitative mass spectrometric analysis of peptide or protein mixtures.     

Microfluidic platform for liquid chromatography-tandem mass spectrometry analyses of complex peptide mixtures

A microfluidic chip that integrates all the fluidic components of a gradient liquid chromatography (LC) system is described. These chips were batch-fabricated on a silicon wafer using photolithographic processes and with Parylene as the main structural material. The fabricated chip includes three electrolysis-based electrochemical pumps, one for loading the sample and the other two for delivering the solvent gradient; platinum electrodes for delivering current to the pumps and establishing the electrospray potential; a low-volume static mixer; a column packed with silica-based reversed-phase support; integrated frits for bead capture; and an electrospray nozzle. The fabricated structures were able to withstand pressures in excess of 250 psi. The device was used to perform a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a mixture of peptides from the trypsin digestion of bovine serum albumen (BSA). Gradient elution through the 1.2-cm column was performed at a flow rate of 80 nL/min. Compared to the analysis of the same sample using a commercial nanoflow LC system, the chromatographic resolution was nearly as good, and the total cycle time was significantly reduced because of the minimal volume between the pumps and the column. Results demonstrate the potential of mass-produced, low-cost microfluidic systems capable of performing LC separations for proteomics applications.     

Ultrafast quantitative 2D NMR: an efficient tool for the measurement of specific isotopic enrichments in complex biological mixtures

Two-dimensional nuclear magnetic resonance (2D NMR) is a promising tool for studying metabolic fluxes by measuring (13)C-enrichments in complex mixtures of (13)C-labeled metabolites. However, the methods reported so far are hampered by very long acquisition durations limiting the use of 2D NMR as a quantitative tool for fluxomics. In this paper, we propose a new approach for measuring specific (13)C-enrichments in a very fast way, by using new experiments based on ultrafast 2D NMR. Two homonuclear 2D experiments (ultrafast COSY and zTOCSY) are proposed to measure (13)C-enrichments in a single scan. Their advantages and limitations are discussed, and their high analytical potentialities are highlighted. Both methods are characterized by an accuracy of 1-2%, an average precision of 3%, and an excellent linearity. The analytical performance is equivalent or better than any of the conventional methods previously reported. The two ultrafast experiments are applied to the measurement of (13)C-enrichments on a biomass hydrolyzate, showing the first known application of ultrafast 2D NMR to a real biological extract. The experiment duration is divided by 200 compared to the conventional methods, while preserving 80% of the quantitative information. This new approach opens new perspectives of application for fluxomics and metabonomics.     

Flexible automated approach for quantitative liquid handling of complex biological samples

A fully automated protein precipitation technique for biological sample preparation has been developed for the quantitation of drugs in various biological matrixes. All liquid handling during sample preparation was automated using a Hamilton MicroLab Star Robotic workstation, which included the preparation of standards and controls from a Watson laboratory information management system generated work list, shaking of 96-well plates, and vacuum application. Processing time is less than 30 s per sample or approximately 45 min per 96-well plate, which is then immediately ready for injection onto an LC-MS/MS system. An overview of the process workflow is discussed, including the software development. Validation data are also provided, including specific liquid class data as well as comparative data of automated vs manual preparation using both quality controls and actual sample data. The efficiencies gained from this automated approach are described.     

Chemicalome and metabolome matching approach to elucidating biological metabolic networks of complex mixtures

Global metabolite identification of complex compound mixtures in biological systems is a very challenging task. Herein, we developed and validated a chemicalome to metabolome matching approach by taking herbal medicine as an example to delineate the metabolic networks of complex systems. This approach consists of five steps of data processing including raw data output, endogenous background subtraction, parent compound and metabolite differentiation, chemicalome to metabolome correlation, and the final validation via manual fragment comparison. Chemicalome to metabolome correlation, the core step of this approach, was performed based on matching the accurate mass differences of pseudomolecular ions between them with the accurate mass changes of known metabolic pathways and validating the matches by validation ions. A step-forward approach that confers a gradual identification of metabolites generated from different steps (1-4) and types (degradation, phase I/II, or mixed) of metabolic reactions was further proposed for chemicalome to metabolome matching. This approach was validated to be very useful and powerful for the metabolite identification of a single compound, a homologous compound mixture, and a complex herbal system. Using this approach, all metabolites (162) detected from urine samples of rats treated with Mai-Luo-Ning injection could be linked to their respective parent compounds, and 143 of them were supported by the final validation via manual fragment analysis. In most cases, more than 80% of the automatic matching results could be supported by the manual fragment validations. A complex metabolic network showing all the possible links between precursors and metabolites was successfully constructed. This study provides a generally applicable approach to global metabolite identification of complex compound mixtures in complex matrixes.     

Selective enrichment of low-abundance peptides in complex mixtures by elution-modified displacement chromatography and their identification by electrospray ionization mass spectrometry

Trace components were selectively enriched and detected in the tryptic digest of recombinant human growth hormone using elution-modified displacement chromatography, a hybrid technique combining features of elution and displacement chromatography. Based on the retention behavior of sample components in the elution mode, rapid and selective trace enrichment and high-resolution separation was achieved in a single step by utilizing appropriate combinations of an eluent such as aqueous acetonitrile with the displacer. Mass spectral and chromatographic analysis of displacement zones revealed up to 400-fold enhancement of the concentration of some low-abundance sample components. Potential application of this technique in proteomics to augment the sensitivity of LC-MS and 2-D gel electrophoretic approaches for the detection of biologically important low-abundance species is discussed.     

Phosphoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line.     

High-resolution capillary isoelectric focusing of complex protein mixtures from lysates of microorganisms

High-resolution capillary isoelectric focusing separations of complex protein mixtures have been obtained for cellular lysates of Saccharomyces cerevisiae, Eschericia coli, and Deinococcus radiodurans. High quality separations are shown to be achievable for total protein concentrations of < 0.1 mg/mL. The separation reproducibility was examined, and the influence of the capillary inner wall coating on resolution investigated using fusedsilica capillaries coated with various hydrophilic polymers including hydroxypropyl cellulose, poly(vinyl alcohol), and linear polyacrylamide. Proteins having an isoelectric point (pI) difference of 0.004 are shown to be separated using a linear carrier ampholyte (linear pH gradient between two electrodes) of 3-10. Approximately 45 discrete peaks in the pI range of 5-7 were obtained for S. cerevisiae, approximately 80 peaks in the pI range of 4.5-8.5 for E. coli, and approximately 210 peaks in the pI range of 3-8.8 for D. radiodurans.     

Genomic and proteomic identification of a DNA-binding protein used in the "fingerprinting" of campylobacter species and strains by MALDI-TOF-MS protein biomarker analysis

We have identified a prominent approximately 10-kDa protein biomarker observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of five thermophilic species of Campylobacter: jejuni, coli, lari, upsaliensis, and helveticus. The biomarker was unambiguously identified by genomic and proteomic sequencing as a DNA-binding protein HU. We report the amino acid sequence of HU as determined by sequencing the hup gene of four species (12 strains): C. jejuni (2), C. coli (4), C. upsaliensis (4) and C. lari(2). Confirmation of the amino acid sequence was obtained by nanoflow high-performance liquid chromatography-tandem mass spectrometry of the tryptic peptides of the extracted/digested HU protein. Protein identification was also confirmed by comparison of the molecular weight (MW) predicted from the hup gene and the MW of HU as measured by high-resolution mass spectrometry. We found the HU protein to be particularly useful as a biomarker in that it strongly ionizes by MALDI and its MW varies between species and among strains within a species. Intra- and interspecies variation of the HU MW is due to changes in the amino acid sequence of the HU protein and not due to co- or posttranslational modifications. The strong ionization efficiency of HU by MALDI is likely due, in part, to four lysine residues clustered at the carboxyl end of the protein. We also report identification of the HU protein biomarker for a C. helveticus strain, whose hup gene was not sequenced, but whose HU amino acid sequence was partially conserved in C. upsaliensis strains. We have also tentatively assigned a approximately 10.5-kDa protein biomarker of a C. concisus strain as an HU protein.     

Integrated microanalytical technology enabling rapid and automated protein identification

Protein identification through peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a standard technique, used in many laboratories around the world. The traditional methodology often includes long incubations (6-24 h) and extensive manual steps. In an effort to address this, an integrated microanalytical platform has been developed for automated identification of proteins. The silicon micromachined analytical tools, i.e., the microchip immobilized enzyme reactor (mu-chip IMER), the piezoelectric microdispenser, and the high-density nanovial target plates, are the cornerstones in the system. The mu-chip IMER provides on-line enzymatic digestion of protein samples (1 microL) within 1-3 min, and the microdispenser enables subsequent on-line picoliter sample preparation in a high-density format. Interfaced to automated MALDI-TOF MS, these tools compose a highly efficient platform that can analyze 100 protein samples in 3.5 h. Kinetic studies on the microreactors are reported as well as the operation of this microanalytical platform for protein identification, wherein lysozyme, myoglobin, ribonuclease A, and cytochrome c have been identified with a high sequence coverage (50-100%).     

Methodology utilizing MS signal intensity and LC retention time for quantitative analysis and precursor ion selection in proteomic LC-MALDI analyses

This study describes a methodology for performing relative quantitation in large-scale proteomic sample comparisons using an LC-MALDI mass spectrometry analytical platform without the use of isotope tagging reagents. The method utilizes replicate analyses of a sample to create a profile of constituent components that are aligned based on LC elution time and mass. Once components from individual runs have been grouped as common "features", the Student's t test is used to determine which components are systematically different between samples. In this study, five HPLC runs of human plasma were compared to five HPLC runs of human serum. About 3889 components were detected in all 10 runs. Of these, 1831 corresponded to approximately 100 known serum proteins, based on MS/MS analysis of one run each from serum and plasma. As expected, fibrinogen alpha, beta, and gamma chains accounted for many of the most significant differences. Therefore, using MALDI, samples containing thousands of peptides can be compared in a minimal amount of time. Moreover, the results of the comparison can be used to guide further MS/MS mode sample interrogation in a result dependent manner.     

使用光纤实现荧光定量PCR检测

阐述了光纤在荧光定量PCR检测中的应用特点和荧光产生机理,给出了光纤耦合效率的计算公式。介绍了试验装置的构成和工作原理,列举了限制荧光检测的实际问题并提供了消除模块背景的方法。试剂检测的结果证实了使用光纤的检测系统具有很高的检测分辨率和8个数量级以上的动态线性范围,完全满足荧光定量PCR检测的要求。     

Peptide mass mapping constrained with stable isotope-tagged peptides for identification of protein mixtures.

Through proteolysis and peptide mass determination using mass spectrometry, a peptide mass map (PMM) can be generated for protein identification. However, insufficient peptide mass accuracy and protein sequence coverage limit the potential of the PMM approach for high-throughput, large-scale analysis of proteins. In our novel approach, nonlabile protons in particular amino acid residues were replaced with deuteriums to mass-tag proteins of the S. cerevisiae proteome in a sequence-specific manner. The resulting mass-tagged proteolytic peptides with characteristic mass-split patterns can be identified in the data search using constraints of both amino acid composition and mass-to-charge ratio. More importantly, the mass-tagged peptides can further act as internal calibrants with high confidence in a PMM to identify the parent proteins at modest mass accuracy and low sequence coverage. As a result, the specificity and accuracy of a PMM was greatly enhanced without the need for peptide sequencing or instrumental improvements to obtain increased mass accuracy. The power of PMM has been extended to the unambiguous identification of multiple proteins in a 1D SDS gel band including the identification of a membrane protein.     

Shift reagents for multidimensional ion mobility spectrometry-mass spectrometry analysis of complex peptide mixtures: evaluation of 18-crown-6 ether complexes

18-Crown-6 ether (18C6) is evaluated as a shift reagent for multidimensional ion mobility spectrometry-mass spectrometry (IMS-IMS-MS) analyses of tryptic protein digests. In this approach, 18C6 is spiked into the solution-phase mixture and noncovalent peptide-crown ion complexes are formed by electrospraying the mixture into the gas phase. After an initial mobility separation in the first IMS drift region, complexes of similar mobility are selected and dissociated via collisional activation prior to entering the second drift region. These dissociation products (including smaller complexes, naked peptide ions, charge transfer products, and fragment ions) differ in mobility from their precursor ion complexes and (in favorable cases) from one another, allowing the mixture to resolve further in the second IMS region. We estimate an IMS-IMS peak capacity of ~2400 when shift reagents are employed. The approach is illustrated by examining a tryptic digest of cytochrome c and by identifying a peptide out of a complex mixture obtained by digestion of human plasma proteins. Disadvantages arising from increased complexity of data sets as well as other advantages of this approach are considered.