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Development and evaluation of normalization methods for label-free relative quantification of endogenous peptides.

ABSTRACT: The performances of 10 different normalization methods on data of endogenous brain peptides produced with label-free nano-LC-MS were evaluated. Data sets originating from three different species (mouse, rat, and Japanese quail), each consisting of 35-45 individual LC-MS analyses, were used in the study. Each sample set contained both technical and biological replicates, and the LC-MS analyses were performed in a randomized block fashion. Peptides in all three data sets were found to display LC-MS analysis order-dependent bias. Global normalization methods will only to some extent correct this type of bias. Only the novel normalization procedure RegrRun (linear regression followed by analysis order normalization) corrected for this type of bias. The RegrRun procedure performed the best of the normalization methods tested and decreased the median S.D. by 43% on average compared with raw data. This method also produced the smallest fraction of peptides with interblock differences while producing the largest fraction of differentially expressed peaks between treatment groups in all three data sets. Linear regression normalization (Regr) performed second best and decreased median S.D. by 38% on average compared with raw data. All other examined methods reduced median S.D. by 20-30% on average compared with raw data.

SUBMITTER: Kultima K

PROVIDER: S-EPMC2758756 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Development and evaluation of normalization methods for label-free relative quantification of endogenous peptides.

Kultima Kim K Nilsson Anna A Scholz Birger B Rossbach Uwe L UL Fälth Maria M Andrén Per E PE

Molecular & cellular proteomics : MCP 20090712 10

The performances of 10 different normalization methods on data of endogenous brain peptides produced with label-free nano-LC-MS were evaluated. Data sets originating from three different species (mouse, rat, and Japanese quail), each consisting of 35-45 individual LC-MS analyses, were used in the study. Each sample set contained both technical and biological replicates, and the LC-MS analyses were performed in a randomized block fashion. Peptides in all three data sets were found to display LC-M ...[more]

PMID: 19596695

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Project description:Overexpressing novel antimicrobial peptides (AMPs) in plants is a promising approach for crop disease resistance engineering. However, the in planta stability and subcellular localization of each AMP should be validated for the respective plant species, which can be challenging due to the small sizes and extreme pI ranges of AMPs which limits the utility of standard proteomic gel-based methods. Despite recent advances in quantitative shotgun proteomics, its potential for AMP analysis has not been utilized and high throughput methods are still lacking.We created transgenic Nicotiana attenuata plants that independently express 10 different AMPs under a constitutive 35S promoter and compared the extracellular accumulation of each AMP using a universal and versatile protein quantification method. We coupled a rapid apoplastic peptide extraction with label-free protein quantification by nanoUPLC-MSE analysis using Hi3 method and identified/quantified 7 of 10 expressed AMPs in the transgenic plants ranging from 37 to 91 amino acids in length. The quantitative comparison among the transgenic plant lines showed that three particular peptides, belonging to the defensin, knottin and lipid-transfer protein families, attained the highest concentrations of 91 to 254 pmol per g leaf fresh mass, which identified them as best suited for ectopic expression in N. attenuata. The chosen mass spectrometric approach proved to be highly sensitive in the detection of different AMP types and exhibited the high level of analytical reproducibility required for label-free quantitative measurements along with a simple protocol required for the sample preparation.Heterologous expression of AMPs in plants can result in highly variable and non-predictable peptide amounts and we present a universal quantitative method to confirm peptide stability and extracellular deposition. The method allows for the rapid quantification of apoplastic peptides without cumbersome and time-consuming purification or chromatographic steps and can be easily adapted to other plant species.

Dataset Information

Development and evaluation of normalization methods for label-free relative quantification of endogenous peptides.

Publications

Development and evaluation of normalization methods for label-free relative quantification of endogenous peptides.

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