Dataset Information

Comparison of protein expression ratios observed by sixplex and duplex TMT labeling method.

ABSTRACT: Stable isotope labeling via isobaric derivatization of peptides is a universally applicable approach that enables concurrent identification and quantification of proteins in different samples using tandem mass spectrometry. In this study, we evaluated the performance of amine-reactive isobaric tandem mass tag (TMT), available as duplex and sixplex sets, with regard to their ability to elucidate protein expression changes. Using rat brain tissue from two different developmental time points, postnatal day 1 (p1) and 45 (p45), as a model system, we compared the protein expression ratios (p45/p1) observed using duplex TMT tags in triplicate measurements versus sixplex tag in a single LC-MS/MS analysis. A correlation of 0.79 in relative protein abundance was observed in the proteins quantified by these two sets of reagents. However, more proteins passed the criteria for significant fold change (-1.0 ? log(2) ratio (p45/p1) ? +1.0 and p < 0.05) in the sixplex analysis. Nevertheless, in both methods most proteins showing significant fold change were identified by multiple spectra, increasing their quantification precision. Additionally, the fold change in p45 rats against p1, observed in TMT experiments, was corroborated by a metabolic labeling strategy where relative quantification of differentially expressed proteins was obtained using (15)N-labeled p45 rats as an internal standard.

SUBMITTER: Rauniyar N

PROVIDER: S-EPMC3751587 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Comparison of protein expression ratios observed by sixplex and duplex TMT labeling method.

Rauniyar Navin N Gao Benbo B McClatchy Daniel B DB Yates John R JR

Journal of proteome research 20121224 2

Stable isotope labeling via isobaric derivatization of peptides is a universally applicable approach that enables concurrent identification and quantification of proteins in different samples using tandem mass spectrometry. In this study, we evaluated the performance of amine-reactive isobaric tandem mass tag (TMT), available as duplex and sixplex sets, with regard to their ability to elucidate protein expression changes. Using rat brain tissue from two different developmental time points, postn ...[more]

PMID: 23214967

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Project description:BACKGROUND:Ramet propagation in strawberry (Fragaria × ananassa) is the most effective way in production. However, the lack of systematically phenotypic observations and high-throughput methods limits our ability to analyze the key factors regulating the heterogeneity in strawberry stolon buds. RESULTS:From observation, we found that the axillary bud located in the first node quickly stepped into dormancy (DSB), after several bract and leaf buds were differentiated. The stolon apical meristem (SAM) degenerated as the new ramet leaf buds (RLB), and the new active axillary stolon buds (ASB) differentiated continually after the differentiation of the first leaf. Using the tandem mass tags (TMT) labeling method, a total of 7271 strawberry proteins were identified. Between ASB and DSB, the spliceosome DEPs, such as Ser/Arg-rich (SR) and heterogeneous nuclear ribonucleoprotein particle (hnRNP), showed the highest enrichment and high PPI connectivity. This indicated that the differences in DEPs (e.g., SF-3A and PK) at the transcriptional level may be causing the differences between the physiological statuses of ASB and DSB. As expected, the photosynthetic pre-form RLB mainly differentiated from ASB and DSB judging by the DEP enrichment of photosynthesis. However, there are still other specialized features of DEPs between RLB and DSB and between ASB and DSB. The DEPs relative to DNA duplication [e.g., minichromosome maintenance protein (MCM 2, 3, 4, 7)], provide a strong hint of functional gene duplication leading the bud heterogeneity between RLB and DSB. In addition, the top fold change DEP of LSH 10-like might be involved in the degeneration of SAM into RLBs, based on its significant function in modulating the plant shoot initiation. As for RLB/ASB, the phenylpropanoid biosynthesis pathway probably regulates the ramet axillary bud specialization, and further promotes the differentiation of xylem when ASB develops into a new stolon [e.g., cinnamyl alcohol dehydrogenase 1 (CAD1) and phenylalanine ammonia-lyase 1 (PAL1)]. CONCLUSIONS:By using phenotypic observation combined with proteomic networks with different types of strawberry stolon buds, the definite dormancy phase of DSB was identified, and the biological pathways and gene networks that might be responsible for heterogeneity among different stolon buds in strawberry were also revealed.

Dataset Information

Comparison of protein expression ratios observed by sixplex and duplex TMT labeling method.

Publications

Comparison of protein expression ratios observed by sixplex and duplex TMT labeling method.

Similar Datasets

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