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MSstats splitplot method for protein quantitation in MS datasets

ABSTRACT: Thirty commercial proteins were prepared at 1.5 pmol/µL in three different subsets of 10 proteins each. Proteins from these subsets were spiked to a 15-µg Escherichia coli background in different amounts and five different mixtures were prepared in triplicates. The final amount of each protein in each mixture was either 100, 200 or 400 fmol/µg of E. coli background for the subsets marked as 1, 2 and 4, respectively. The generated dataset with known concentrations of spiked-in proteins was used to evaluate the performance of several processing tools of MS proteomics datasets.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Escherichia Coli (ncbitaxon:562)

SUBMITTER: Eduard Sabid�

PROVIDER: MSV000084181 | MassIVE | Thu Aug 08 07:39:00 BST 2019

SECONDARY ACCESSION(S): PXD005642

REPOSITORIES: MassIVE

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Project description:Being inspired by metabolomic data processing, we have developed a bioinformatic pipeline that optimizes the processing of mass spectral data obtained from isobaric Tandem Mass Tag (TMT) experiments. Our method focuses on the tandem mass spectral level by first quantifying and then identifying (QtI), while preserving unidentified spectra for further investigations. The raw datasets were previously generated [1, 2]. Two-proteome model experiments were considered where identical pools of human CSF or plasma samples were mixed with E. coli samples at different concentrations. E. coli protein extract was spiked in 400 µL of CSF at amounts of 0, 2, 3, 5, 6.25, and 7.5 µg. Such sets of 6 spiked CSF samples were prepared in triplicate for comparison using sixplex isobaric tagging and analyzed in triplicates on two independent but identical LC MS/MS, for a total of 18 raw files [1]; this experiment is called “CSF-E.coli”. E. coli protein extract was spiked at 0, 2.5, 5, 6.25, 12.5, and 25 µg in 30 µL human plasma. Such sets of 6 spiked plasma samples were prepared in quadruplicate for comparison using sixplex isobaric tagging and analyzed in triplicates on one LC MS/MS, for a total of 12 raw files [2]; this experiment is referred as “Plasma-E.coli”.The so-called “96samples-CSF” experiment consists of 16 replicate TMT sixplex experiments measuring identical CSF samples from the pool described above [2], analyzed in triplicates on one LCMS/MS for a total of 48 raw files. The so-called “96samples-plasma” experiment consists of 16 replicate TMT sixplex experiments measuring identical plasma samples from the pool described before, analyzed in triplicates on one LC MS/MS for a total of 48 raw files [1]. References: [1] Dayon, L., Núñez Galindo, A., Corthésy, J., Cominetti, O. & Kussmann, M. Comprehensive and scalable highly automated MS-based proteomic workflow for clinical biomarker discovery in human plasma. J. Proteome Res. 13, 3837-3845 (2014). [2] Núñez Galindo, A., Kussmann, M. & Dayon, L. Proteomics of Cerebrospinal Fluid: Throughput and Robustness Using a Scalable Automated Analysis Pipeline for Biomarker Discovery. Anal. Chem. 87, 10755-10761 (2015).

Project description:Methods: During the last trimester of the gestation, 12 heifers were immunized using intramuscular injection with heat-killed E. coli P4 emulsified in Montanide ISA 61VG® oil adjuvant (SEPPIC), as recommended by the manufacturer. Two months later, half of the heifers (IM group, n=6) got a second injection as a booster. Others received 50 µg of protein concentrate prepared from E. coli P4 supernate in two quarters (IMM group, n=6). Cows of the control group (CONT, n=6) were injected intramuscularly with adjuvant only at the same dates. Cows were then challenged on average at 49 days in milk in one healthy quarter by infusion of a E. coli P4 bacterial suspension (10e3 bacteria). Blood was collected before experiment, and seven days after the first and the second injection (immunization phase), and then at 0, 12 and 40 hours after infection (challenge phase). Results : Whole blood transcriptome through RNA-seq identifies the main biological functions altered during an E. coli mastitis. Systemic and mammary routes of E. coli immunization elicit different gene expression profiles that correlated with the levels of protection Conclusions: Intramuscular immunization is correlated with a high systemic Th1 response that is not as protective as the local route of immunization. During the last trimester of the gestation, 12 heifers were immunized using intramuscular injection with heat-killed E. coli P4 emulsified in Montanide ISA 61VG® oil adjuvant (SEPPIC), as recommended by the manufacturer. Two months later, half of the heifers (IM group, n=6) got a second injection as a booster. Others received 50 µg of protein concentrate prepared from E. coli P4 supernate in two quarters (IMM group, n=6). Cows of the control group (CONT, n=6) were injected intramuscularly with adjuvant only at the same dates. Cows were then challenged on average at 49 days in milk in one healthy quarter by infusion of a E. coli P4 bacterial suspension (10e3 bacteria). Blood was collected before experiment, and seven days after the first and the second injection (immunization phase), and then at 0, 12 and 40 hours after infection (challenge phase).

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MSstats splitplot method for protein quantitation in MS datasets

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