Dataset Information

Improving Mass Spectrometer Acquisition when using Tandem MS/MS/MS Scanning with Isobaric Tags

ABSTRACT: n this work we utilize a standard, high-complexity Human cell line tryptic digest combined with a synthetic peptide library to systematically evaluate the properties of a variety of MS operation modes and detector configurations for the optimal examination of isobaric-tagged peptide mixtures. We demonstrate that despite the ability of the IT to yield more rapid MS3 scanning, the benefit in peptide and protein identifications when examining complex mixtures is minor while the negative impact on quantification precision is significant.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Yarrowia Lipolytica (candida Lipolytica) Homo Sapiens (human) Ralstonia Eutropha Jmp134

SUBMITTER: Christopher Hughes

LAB HEAD: Gregg Morin

PROVIDER: PXD005890 | Pride | 2017-04-26

REPOSITORIES: Pride

ACCESS DATA

Publications

Investigating Acquisition Performance on the Orbitrap Fusion When Using Tandem MS/MS/MS Scanning with Isobaric Tags.

Hughes Christopher S CS Spicer Victor V Krokhin Oleg V OV Morin Gregg B GB

Journal of proteome research 20170420 5

Methods for isobaric-tagged peptide analysis (e.g., TMT, iTRAQ), such as the synchronous precursor selection (SPS) tandem MS/MS/MS (MS<sup>3</sup>) approach, enable maintenance of reporter ion accuracy and precision by reducing the ratio compression caused by coisolated precursor ions. However, the decreased throughput of the MS<sup>3</sup> approach necessitates careful optimization of acquisition strategies and methods to ensure maximal proteome coverage. We present a systematic analysis of acq ...[more]

PMID: 28418257

Similar Datasets

Project description:Modification of microRNA sequences by the 3' addition of nucleotides to generate so-called M-bM-^@M-^\isomiRsM-bM-^@M-^] adds to the complexity of miRNA function, with recent reports showing that 3' modifications can influence miRNA stability and efficiency of target repression. Here we show that the 3' modification of miRNAs is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species ranging from C. elegans to human. The modifications result predominantly from adenylation and uridylation, and are seen across tissue types, disease states, and developmental stages. To quantitatively profile 3' nucleotide additions, we developed and validated a novel assay based on NanoString Technologies' nCounter platform. For certain miRNAs, the frequency of modification was altered by processes such as cell differentiation, indicating that 3' modification is a biologically regulated process. To investigate the mechanism of 3' nucleotide additions, we used RNA interference to screen a panel of eight candidate miRNA nucleotidyl transferases for 3' miRNA modification activity in human cells. Multiple enzymes, including PAPD1, PAPD4, PAPD5, ZCCHC6, ZCCHC11, and TUT1, were found to govern 3' nucleotide addition to miRNAs in a miRNA-specific manner. Three of these enzymesM-bM-^@M-^SPAPD1, ZCCHC6 and TUT1M-bM-^@M-^Shave not previously been known to modify miRNAs. Collectively, our results indicate that 3' modification observed in next generation small RNA sequencing data is a biologically relevant process, and identify enzymatic mechanisms that may lead to new approaches for modulating miRNA activity in vivo. We validated the specificity of the platform by assaying three pools of synthetic RNA oligonucleotides representing canonical, variant 1, and variant 2 versions of 5 miRNAs (miR-15a, miR-15b, miR-125a-5p, miR-143 and miR-221).Each bridge pool (i.e., specific to canonical, variant 1 or variant 2) was used to assay the three mixtures of synthetic miRNA oligonucleotides. In order to assess accuracy, we also assayed mixtures of 5 synthetic miRNAs containing 60% canonical miRNA, 30% variant 1, and 10% variant 2. Triplicate technical replicates of each sample were performed.

Dataset Information

Improving Mass Spectrometer Acquisition when using Tandem MS/MS/MS Scanning with Isobaric Tags

Publications

Investigating Acquisition Performance on the Orbitrap Fusion When Using Tandem MS/MS/MS Scanning with Isobaric Tags.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure