Automated 2D Peptide Separation on a 1D Nano-LC-MS System

Taylor, Paul; Nielsen, Per Mose; Trelle, Morten Beck; Hørning, Ole; Andersen, Michael Brun; Vorm, Ole; Moran, Michael F.; Kislinger, Thomas

doi:10.1021/pr800986c

Cited by 58 publications

(72 citation statements)

References 21 publications

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“…For peptide separation on the analytical column, a water/acetonitrile gradient, controlled by the EASY-nLC (Thermo Scientific), was applied at an effective flow rate of 400 nL/min. Ammonium acetate salt bumps (8 l) at concentrations of 100, 150, 200, and 500 mM were sequentially loaded, and peptides were eluted by a water/acetonitrile gradient as described previously (22). Sample analysis was performed on an LTQ Orbitrap XL (Thermo Scientific) using previously described instrument parameters (22).…”

Section: Methodsmentioning

confidence: 99%

Integrated Omic Analysis of Oropharyngeal Carcinomas Reveals Human Papillomavirus (HPV)–dependent Regulation of the Activator Protein 1 (AP-1) Pathway

Sepiashvili

Hui

Shi

et al. 2014

Molecular & Cellular Proteomics

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a HPV-positive oropharyngeal carcinoma (OPC) patients have superior outcomes relative to HPV-negative patients, but the underlying mechanisms remain poorly understood. We conducted a proteomic investigation of HPV-positive (n ‫؍‬ 27) and HPV-negative (n ‫؍‬ 26) formalinfixed paraffin-embedded OPC biopsies to acquire insights into the biological pathways that correlate with clinical behavior. Among the 2,633 proteins identified, 174 were differentially abundant. These were enriched for proteins related to cell cycle, DNA replication, apoptosis, and immune response. The differential abundances of cortactin and methylthioadenosine phosphorylase were validated by immunohistochemistry in an independent cohort of 29 OPC samples (p ‫؍‬ 0.023 and p ‫؍‬ 0.009, respectively). An additional 1,124 proteins were independently corroborated through comparison to a published proteomic dataset of OPC. Furthermore, utilizing the Cancer Genome Atlas, we conducted an integrated investigation of OPC, attributing mechanisms underlying differential protein abundances to alterations in mutation, copy number, methylation, and mRNA profiles. A key finding of this integration was the identification of elevated cortactin oncoprotein levels in HPV-negative OPCs. These proteins might contribute to reduced survival in these patients via their established role in radiation resistance. Through interrogation of Cancer Genome Atlas data, we demonstrated that activation of the ␤1-integrin/FAK/cortactin/

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Section: Methodsmentioning

confidence: 99%

Integrated Omic Analysis of Oropharyngeal Carcinomas Reveals Human Papillomavirus (HPV)–dependent Regulation of the Activator Protein 1 (AP-1) Pathway

Sepiashvili

Hui

Shi

et al. 2014

Molecular & Cellular Proteomics

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“…MudPIT Analysis of HNSCC Cell Lines-A fully automated 5-cycle two-dimensional chromatography sequence was set up as previously described (22). Peptides were loaded on a 7 cm precolumn (150 m i.d.)…”

Section: Methodsmentioning

confidence: 99%

“…In the current study, we focused on multidimensional protein identification technology (MudPIT) analyses of secretomes and whole cell lysate proteomes of three head and neck cancer (HNC) cell lines: FaDu, UTSCC8, and UTSCC42a, coupled with parallel gene expression analyses, as a platform for discovery of putative secreted HNSCC markers (21,22). These cell lines were derived from two anatomically related subsites of the head and neck: the hypopharynx (FaDu cells) and the larynx (UTSCC8, and UTSCC42a cells).…”

Section: Head and Neck Squamous Cell Carcinoma (Hnscc)mentioning

confidence: 99%

Potentially Novel Candidate Biomarkers for Head and Neck Squamous Cell Carcinoma Identified Using an Integrated Cell Line-based Discovery Strategy

Sepiashvili

Hui

Ignatchenko

et al. 2012

Molecular & Cellular Proteomics

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Head and neck squamous cell carcinomas (HNSCC) can arise from the oral cavity, oropharynx, larynx or hypopharynx, and is the sixth leading cancer by incidence worldwide. The 5-year survival rate of HNSCC patients remains static at 40 -60%. Hence, biomarkers which can improve detection of HNSCC or early recurrences should improve clinical outcome. Mass spectrometry-based proteomics methods have emerged as promising approaches for biomarker discovery. As one approach, mass-spectrometric identification of proteins shed or secreted from cancer cells can contribute to the identification of potential biomarkers for HNSCC and our understanding of tumor behavior. In the current study, mass spectrometry-based proteomic profiling was performed on the conditioned media (i.e. secretome) of head and neck cancer (HNC) cell lines (FaDu, UTSCC8 and UTSCC42a) in addition to gene expression microarrays to identify over-expressed transcripts in the HNSCC cells in comparison to a normal control cell line. This integrated data set was systematically mined using publicly available resources (Human Protein Atlas and published proteomic/transcriptomic data) to prioritize putative candidates for validation. Subsequently, quantitative real-time PCR (qRT-PCR), Western blotting, immunohistochemistry (IHC), and ELISAs were performed to verify selected markers. Our integrated analyses identified 90 putative protein biomarkers that were secreted or shed to the extracellular space and over-expressed in HNSCC cell lines, relative to controls. Subsequently, the over-expression of five markers was verified in vitro at the transcriptional and translational levels using qRT-PCR and Western blotting, respectively. IHC-based validation conducted in two independent Head and Neck Squamous Cell Carcinoma (HNSCC)1 is the sixth most common cancer worldwide, with ϳ600,000 new cases diagnosed every year (1). HNSCCs include squamous cell carcinomas (SCCs) of the oral cavity, pharynx (nasopharynx, oropharynx, and hypopharynx), and larynx. Despite improvements in therapeutic approaches, the overall 5-year survival rate of HNSCC patients has not improved over the past three decades (1, 2).Clinical management of HNSCC is faced by several challenges, including early detection of the primary tumor, and site-specific control of the disease (3, 4). Early diagnosis is often hampered by the asymptomatic nature of HNSCC development and the inadequacy of current diagnostic methods to identify HNSCCs with sufficient specificity and sensitivity. As a result, over 60% of HNSCC patients present with advanced stages III or IV, harboring lymph node metastases (3).

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“…To assess the contribution of the NB TIC-enriched genes to the NB TIC proteome, we conducted a MudPIT analysis (14) of whole-cell lysate and a membrane-enriched fraction of NB TIC line NB88R2 generated from a bone marrow metastasis of a high-risk patient. The MudPIT approach effectively identifies hundreds to thousands of concurrently expressed proteins for global or subcellular fraction-specific proteomic profile analyses (24).…”

Section: Comparison Of Transcripts Differentially Abundant In Nb Ticsmentioning

confidence: 99%

System-Level Analysis of Neuroblastoma Tumor–Initiating Cells Implicates AURKB as a Novel Drug Target for Neuroblastoma

Morozova

Vojvodic

Grinshtein

et al. 2010

Clinical Cancer Research

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Purpose: Neuroblastoma (NB) is an aggressive tumor of the developing peripheral nervous system that remains difficult to cure in the advanced stages. The poor prognosis for high-risk NB patients is associated with common disease recurrences that fail to respond to available therapies. NB tumor-initiating cells (TICs), isolated from metastases and primary tumors, may escape treatment and contribute to tumor relapse. New therapies that target the TICs may therefore prevent or treat tumor recurrences.Experimental Design: We undertook a system-level characterization of NB TICs to identify potential drug targets against recurrent NB. We used next-generation RNA sequencing and/or human exon arrays to profile the transcriptomes of 11 NB TIC lines from six NB patients, revealing genes that are highly expressed in the TICs compared with normal neural crest-like cells and unrelated cancer tissues. We used gel-free two-dimensional liquid chromatography coupled to shotgun tandem mass spectrometry to confirm the presence of proteins corresponding to the most abundant TIC-enriched transcripts, thereby providing validation to the gene expression result.Results: Our study revealed that genes in the BRCA1 signaling pathway are frequently misexpressed in NB TICs and implicated Aurora B kinase as a potential drug target for NB therapy. Treatment with a selective AURKB inhibitor was cytotoxic to NB TICs but not to the normal neural crest-like cells.Conclusion: This work provides the first high-resolution system-level analysis of the transcriptomes of 11 primary human NB TICs and identifies a set of candidate NB TIC-enriched transcripts for further development as therapeutic targets. Clin Cancer Res; 16(18); 4572-82. ©2010 AACR.

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Automated 2D Peptide Separation on a 1D Nano-LC-MS System

Cited by 58 publications

References 21 publications

Integrated Omic Analysis of Oropharyngeal Carcinomas Reveals Human Papillomavirus (HPV)–dependent Regulation of the Activator Protein 1 (AP-1) Pathway

Integrated Omic Analysis of Oropharyngeal Carcinomas Reveals Human Papillomavirus (HPV)–dependent Regulation of the Activator Protein 1 (AP-1) Pathway

Potentially Novel Candidate Biomarkers for Head and Neck Squamous Cell Carcinoma Identified Using an Integrated Cell Line-based Discovery Strategy

System-Level Analysis of Neuroblastoma Tumor–Initiating Cells Implicates AURKB as a Novel Drug Target for Neuroblastoma

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