EasyProt — An easy-to-use graphical platform for proteomics data analysis

Gluck, Florent; Hoogland, Christine; Antinori, Paola; Robin, Xavier; Nikitin, Frédéric; Zufferey, Anne; Pasquarello, Carla; Fétaud, Vanessa; Dayon, Loı̈c; Müller, Markus; Lisacek, Frédérique; Geiser, Laurent; Hochstrasser, Denis F.; Sanchez, Jean‐Charles; Scherl, Alexander

doi:10.1016/j.jprot.2012.12.012

Cited by 58 publications

(68 citation statements)

References 21 publications

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“…A maximum of eight precursor ions were selected for collision-induced dissociation (CID) in the LTQ analyzer with a dynamic exclusion time set to 45 s. Ions were accumulated to a target value of 7 ϫ 10 3 with an isolation width of 2 m/z and CID was performed at a normalized collision energy of 35%. Peak lists were generated from raw data using the embedded software from the instrument vendor (extract_MSN.exe) and were subsequently submitted to EasyProt (v2.2), a platform that uses Phenyx (GeneBio, Geneva, Switzerland) for protein identification (27). Searches were conducted against the UniProtKB/SwissProt database (11.01.2011 and 13.06.2012 releases containing 20Ј252 and 20Ј237 reviewed entries respectively) specifying "Homo sapiens" taxonomy.…”

Section: Methodsmentioning

confidence: 99%

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues

Steiner

Tille

Lamerz

et al. 2015

Molecular & Cellular Proteomics

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The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selected reaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R 2 : 0.99 -1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website. MS based proteomics has traditionally been used to investigate complex biological systems, such as cell lines, plasma, or fresh-frozen tissues (1, 2). In the last decade however, MS proteomics has extended to the analysis of formalin-fixed paraffin-embedded (FFPE) 1 tissues (3). Formalin fixation is the gold standard for sample storage in clinical pathology because it allows optimal preservation of the morphological features of the tissue and it is economically attractive (storage at room temperature over several years or decades) (4). Sev-

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Section: Methodsmentioning

confidence: 99%

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues

Steiner

Tille

Lamerz

et al. 2015

Molecular & Cellular Proteomics

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“…After the last wash, His-6-tagged peptides were eluted by incubating the beads in 80 μL of 200 mM imidazole, 0.15 M Tris·HCl (pH 6.7), 30% glycerol, and 0.72 M β-mercaptoethanol for 30 min at room temperature. Peptides were then digested with trypsin before analysis by LC-MS/MS as described previously (33).…”

Section: Methodsmentioning

confidence: 99%

“…Database searches and false-discovery rate estimation were performed using the EasyProt software platform (33). Only peptide spectra matches with a false-discovery rate <1% were retained.…”

Section: Methodsmentioning

confidence: 99%

Translation of pre-spliced RNAs in the nuclear compartment generates peptides for the MHC class I pathway

Apcher

Millot

Daskalogianni

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The scanning of maturing mRNAs by ribosomes plays a key role in the mRNA quality control process. When ribosomes first engage with the newly synthesized mRNA, and if peptides are produced, is unclear, however. Here we show that ribosomal scanning of prespliced mRNAs occurs in the nuclear compartment, and that this event produces peptide substrates for the MHC class I pathway. Inserting antigenic peptide sequences in introns that are spliced out before the mRNAs exit the nuclear compartment results in an equal amount of antigenic peptide products as when the peptides are encoded from the main open reading frame (ORF). Taken together with the detection of intron-encoded nascent peptides and RPS6/RPL7-carrying complexes in the perinucleolar compartment, these results show that peptides are produced by a translation event occurring before mRNA splicing. This suggests that ribosomes occupy and scan mRNAs early in the mRNA maturation process, and suggests a physiological role for nuclear mRNA translation, and also helps explain how the immune system tolerates peptides derived from tissue-specific mRNA splice variants.MHC class I restricted antigen presentation | mRNA maturation | nuclear translation R NAs carrying premature termination codons or are recognized as defective in other aspects are prevented from further translation and disposed of by the nonsense-mediated decay (NMD) pathway (1). The detection of premature stop codons is mediated by the scanning ribosome; however, when the ribosomes first engage with the newly synthesized mRNA, and if this event results in the production of peptides, is unclear. Two observations from the field of immunology and the presentation of peptides on MHC class I molecules have highlighted some aspects relevant to the role of the ribosomes in the mRNA maturation process. The first observation is related to the detection of antigenic peptides originating from intron sequences, and the second is related to the observation that the synthesis of antigenic peptide substrates and full-length proteins are two spatiotemporarily distinct events (2-5).The presentation of antigenic peptides on MHC class I molecules allows CD8 + T cells to detect and eliminate cells in which the repertoire of peptide substrates has been altered owing to the presence of viruses or to changes in the presentation of endogenous antigens (6, 7). However, despite the key role of antigen presentation on MHC class I molecules in immune surveillance, the source of peptides for the MHC class I pathway is not yet known. Accumulating evidence indicates that the processing of alternative peptide substrates plays a major role, and that degradation products derived from full-length proteins have limited access to the MHC class I pathway (8-10). We recently demonstrated that pioneer translation products (PTPs) that form antigenic peptide substrates for the MHC class I pathway are produced by a translation event that is distinct from the canonical event giving rise to full-length proteins and does not require the cap-binding ...

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“…Peak lists (MGF file format) were generated from raw orbitrap data using the EasyProtConv conversion tool (version 1.6) from the EasyProt software platform (17). The peak list files were searched against the SwissProt database (release 15.10 of 21-Sept-2011) using Mascot (version 2.2.0; Matrix Science, Ltd., London, UK).…”

Section: Peptide Fragmentation Sequencing Lc-esi-ms/ms Was Performedmentioning

confidence: 99%

Comparative secretome of ovarian serous carcinoma: Gelsolin in the spotlight

et al. 2017

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Abstract. Ovarian cancer is one of the most common types of reproductive cancer, and has the highest mortality rate amongst gynecological cancer subtypes. The majority of ovarian cancers are diagnosed at an advanced stage, resulting in a five-year survival rate of ~30%. Early diagnosis of ovarian cancer has improved the five-year survival rate to ≥90%, thus the current imperative requirement is to identify biomarkers that would allow the early detection, diagnosis and monitoring of the progression of the disease, or of novel targets for therapy. In the present study, secreted proteins from purified ovarian control, benign and cancer cells were investigated by mass spectrometry, in order to identify novel specific markers that are easy to quantify in patients sera. A total of nine proteins revealed significant differential secretion from control and benign cells, in comparison with ovarian cancer cells. The mRNA expression levels of three of these proteins (Dickkopf protein 3, heat shock protein 10 kDa and gelsolin) were subsequently evaluated by reverse transcription-quantitative polymerase chain reaction. Combined with the protein level in serum, the present study identified that gelsolin may be a useful marker of ovarian cancer.

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EasyProt — An easy-to-use graphical platform for proteomics data analysis

Cited by 58 publications

References 21 publications

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues

Translation of pre-spliced RNAs in the nuclear compartment generates peptides for the MHC class I pathway

Comparative secretome of ovarian serous carcinoma: Gelsolin in the spotlight

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