Systematic Comparison of a Two-dimensional Ion Trap and a Three-dimensional Ion Trap Mass Spectrometer in Proteomics

For the archaeon Methanococcus maripaludis, a fully sequenced and annotated model species of hydrogenotrophic methanogen, we report validation of quantitative protein level expression ratios on a proteome-wide basis. Using an approach based on quantitative multidimensional capillary HPLC and quadrupole ion trap mass spectrometry, coverage of gene expression approached that currently achievable with transcription microarrays. Comprehensive mass spectrometry-based proteomics and spotted cDNA arrays were used to compare global protein and mRNA levels in a wild-type (S2) and mutant strain (S40) of M. maripaludis. Using linear regression with 652 expression ratios generated by both the proteomic and microarray methods, a product moment correlation coefficient of 0.24 was observed. The correlation improved to 0.61 if only genes showing significant expression changes were included. A novel two-stage method of outlier detection was used for the protein measurements when Dixon's Q-test by itself failed to give satisfactory results. The log 2 transformations of the number of peptides or isotopic peptide pairs associated with each ORF, divided by the predicted molecular weight, were found to have moderately positive correlations with two bioinformatic predictors of gene expression based on codon bias. We detected peptides derived from 939 proteins or 55% of the genome coding capacity. Of these, 60 were overexpressed, and 34 were underexpressed in the mutant. Of the 1722 ORFs encoded in the genome, 1597 or 93% were probed by cDNA arrays. Of these, 50 were more highly expressed, and 45 showed lower expression levels in the mutant relative to the wild type. 15 ORFs were shown to be overexpressed by both methods, and two ORFs were shown to be overexpressed by proteomics and underexpressed by microarray. Molecular & Cellular Proteomics 5:, 868 -881.The hydrogenotrophic methanogens are a major group of Archaea that conserve energy from the use of molecular hydrogen or formate to reduce carbon dioxide to methane. Methanococcus maripaludis is a model species of hydrogenotrophic methanogen, having favorable laboratory growth behavior, excellent genetic tools (1), and a fully sequenced genome (2). In another publication (3) we describe the molecular biology of a mutant of M. maripaludis (S40) in which the operon encoding the Ehb hydrogenase was disrupted, and functional changes were observed that led to a model for the role of Ehb in energy metabolism. Here we present validation of the multidimensional capillary HPLC/tandem mass spectrometry approach (also known as MudPIT 1 (4, 5)) to quantitative and qualitative proteomics for M. maripaludis by comparison with mRNA expression. Transcriptome analysis was performed in which the ehb mutant and wild-type strains were compared globally for all known genes under the same culture conditions as for the proteomics. For a subset of genes the proteomic results were further validated by real time quantitative RT-PCR. The relative absence of evidence for posttranscriptional gene regulation and ...

Section: Maripaludis Strains S2 and S40 Genome Sequence For S2 Andsupporting

confidence: 73%

Quantitative Proteomics of the Archaeon Methanococcus maripaludis Validated by Microarray Analysis and Real Time PCR

Xia

Hendrickson

Zhang

et al. 2006

“…4). Thus, 2-D (linear) ion traps with superior trapping efficiency, ion capacity, and duty cycle time compared with the conventional three-dimensional ion trap are applicable for sensitive quantification (30,31).…”

Section: Resultsmentioning

confidence: 99%

“…Quantification by LC-SRM (AQUA)-The setup used for on-line microcapillary LC and microelectrospray was essentially as reported previously (30). The dimension of the fused silica capillary column (Polymicrotechnologies, Inc.) was increased from 11 cm ϫ 100 m to 17 cm ϫ 125 m. Magic C 18 beads (200 Å, 5 m, from Michrom BioResources, Auburn, CA) were used for packing the tapered fused silica tubing.…”

Section: Preparation Of Internal Standardmentioning

confidence: 99%

Absolute Quantification of Multisite Phosphorylation by Selective Reaction Monitoring Mass Spectrometry

Mayya

Rezual

et al. 2006

Self Cite

128

124

Multisite phosphorylation is an important mechanism for achieving intricate regulation of protein function. Here we extended the absolute quantification of abundance (AQUA) methodology and validated its applicability to quantitatively study multisite phosphorylation. As a test case, we chose the conserved inhibitory site of the cyclindependent kinases (CDKs), Cdk1, Cdk2, and Cdk3, which are important regulators of cell cycle transitions and apoptosis. Inhibitory phosphorylation at Thr 14 and Tyr 15 of the CDKs is modulated by complex regulatory mechanisms involving multiple kinases and phosphatases. Yet the resulting quantitative dynamics among the four possible phosphorylated and non-phosphorylated versions of CDKs (T14p-Y15p, T14p-Y15, T14-Y15p, and T14-Y15) has not been investigated to date. Hence we used the heavy isotope-labeled tryptic peptides spanning the inhibitory site as internal standards and quantified all four versions by LC-selected reaction monitoring. Quantification of the phosphorylation status of the inhibitory site in the cell extracts provided novel quantitative insights. 1) The transition to mitotic phase was dominated by the conversion of "T14p-Y15p" to the "T14-Y15" form, whereas the two monophosphorylated forms were considerably lower in abundance.2) The amount of all four forms decreased during the progression of apoptosis but with differing kinetics. Analysis of immunoprecipitated Cdk1 and Cdk2 revealed that the inhibitory site phosphorylation state of both kinases at different stages of the cell cycle followed the same trend. Quantitative immunoblotting using antibodies to Cdk1 and Cdk2 and to the T14-Y15p form suggested that quantification by AQUA was reliable and accurate. These results highlight the utility of internal standard peptides to achieve accurate quantification of multisite phosphorylation status.

“…With the recent development of high speed 2D linear ion trap instruments, i.e. LTQ, the protein profiling coverage has been greatly enhanced compared with traditional three-dimensional ion trap systems (25). When coupled with SCX fractionation either on line or off line (26,27), LC-MS/MS technologies now routinely allow for identification of thousands of proteins from complex mammalian tissues and cells.…”

Section: Advances In Lc-ms Technologiesmentioning

confidence: 99%

Advances and Challenges in Liquid Chromatography-Mass Spectrometry-based Proteomics Profiling for Clinical Applications

Qian

Jacobs

Liu

et al. 2006

330

284

Recent advances in proteomics technologies provide tremendous opportunities for biomarker-related clinical applications; however, the distinctive characteristics of human biofluids such as the high dynamic range in protein abundances and extreme complexity of the proteomes present tremendous challenges. In this review we summarize recent advances in LC-MS-based proteomics profiling and its applications in clinical proteomics as well as discuss the major challenges associated with implementing these technologies for more effective candidate biomarker discovery. Developments in immunoaffinity depletion and various fractionation approaches in combination with substantial improvements in LC-MS platforms have enabled the plasma proteome to be profiled with considerably greater dynamic range of coverage, allowing many proteins at low ng/ml levels to be confidently identified. Despite these significant advances and efforts, major challenges associated with the dynamic range of measurements and extent of proteome coverage, confidence of peptide/protein identifications, quantitation accuracy, analysis throughput, and the robustness of present instrumentation must be addressed before a proteomics profiling platform suitable for efficient clinical applications can be routinely implemented.