Trends in sample preparation for classical and second generation proteomics

Cañas, Benito; Piñeiro, Carmen; Calvo, Enrique; López-Ferrer, Daniel; Gallardo, José Manuel

doi:10.1016/j.chroma.2007.01.045

Cited by 166 publications

(150 citation statements)

References 232 publications

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“…Certain advantages embedded within the workflow developed for this study include the efficient solubilization and digestion of proteins from FFPE archival tissue without fractionation, such that they are amenable for identification using tandem MS for a complete proteomic representation (7). Furthermore, optimization of combining laser capture microdissection with shotgun proteomic technologies enabled the detection of proteins expressed primarily within the tumor cells rather than in the stroma and other complex cellular components of the tumor microenvironment.…”

Section: Discussionmentioning

confidence: 99%

Proteomic Analysis of Laser-Captured Paraffin-Embedded Tissues: A Molecular Portrait of Head and Neck Cancer Progression

Patel

Hood

Molinolo

et al. 2008

Clinical Cancer Research

189

151

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Purpose: Squamous cell carcinoma of the head and neck (HNSCC), the sixth most prevalent cancer among men worldwide, is associated with poor prognosis, which has improved only marginally over the past three decades. A proteomic analysis of HNSCC lesions may help identify novel molecular targets for the early detection, prevention, and treatment of HNSCC. Experimental Design: Laser capture microdissection was combined with recently developed techniques for protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues and a novel proteomics platform. Approximately 20,000 cells procured from FFPE tissue sections of normal oral epithelium and well, moderately, and poorly differentiated HNSCC were processed for mass spectrometry and bioinformatic analysis. Results: A large number of proteins expressed in normal oral epithelium and HNSCC, including cytokeratins, intermediate filaments, differentiation markers, and proteins involved in stem cell maintenance, signal transduction, migration, cell cycle regulation, growth and angiogenesis, matrix degradation, and proteins with tumor suppressive and oncogenic potential, were readily detected. Of interest, the relative expression of many of these molecules followed a distinct pattern in normal squamous epithelia and well, moderately, and poorly differentiated HNSCC tumor tissues. Representative proteins were further validated using immunohistochemical studies in HNSCC tissue sections and tissue microarrays. Conclusions: The ability to combine laser capture microdissection and in-depth proteomic analysis of FFPE tissues provided a wealth of information regarding the nature of the proteins expressed in normal squamous epithelium and during HNSCC progression, which may allow the development of novel biomarkers of diagnostic and prognostic value and the identification of novel targets for therapeutic intervention in HNSCC.

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Section: Discussionmentioning

confidence: 99%

Proteomic Analysis of Laser-Captured Paraffin-Embedded Tissues: A Molecular Portrait of Head and Neck Cancer Progression

Patel

Hood

Molinolo

et al. 2008

Clinical Cancer Research

189

151

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“…To increase the analytical depth in proteomics studies, a vast number of pre-fractionation strategies have been described in the literature [1,2]. In human body fluids, the analytical challenges mainly arise from the high dynamic range of concentrations, where some high-abundant proteins completely dominate the protein content, making transient low-abundant biomarkers extremely hard to detect, particularly in a non-targeted discovery phase [3].…”

Section: Introductionmentioning

confidence: 99%

Use of narrow‐range peptide IEF to improve detection of lung adenocarcinoma markers in plasma and pleural effusion

et al. 2009

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In this study we applied narrow-range peptide IEF to plasma or pleural effusion prior to LC/MS/MS. Two methods for narrow-range IEF were run; IPG strips and free-flow electrophoresis. Data from this study was compared with cell line data to evaluate the method performance in body fluids. To test the methods potential in quantitative biomarker discovery studies, plasma and pleural effusion from patients with lung adenocarcinoma (n 5 3) were compared with inflammatory pleuritis (n 5 3) using iTRAQ quantification. Using narrow-range IEF on the peptide level we were able to identify and quantify 282 proteins in plasma and 300 proteins in pleural effusion. These body fluid proteomes demonstrated high degree of overlap; however, more proteins significantly differently altered levels related to adenenocarcinoma were found in pleural effusion compared with plasma, suggesting enrichment of lung tissue-related proteins in pleural effusion. Nine proteins were chosen for initial validation with Western blot, and one protein (NPC2) was chosen for further validation using imunohistochemistry. Overall, the quantitative results from IEF/LC/MS/MS showed good correlation with the results from Western blot and imunohistochemistry, showing the potential of this methodology in quantitative biomarker discovery studies.

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“…Reversed phase chromatography of biological macromolecules is a promising separation 53 technique in the pharmaceutical field [1,2], for isolation [3,4], in proteomics [5][6][7], and in 54 various related areas [8][9][10][11][12]. In the separation both the stationary phase and mobile phase play Biological macromolecules are typically separated in acidic conditions.…”

mentioning

confidence: 99%

Influence of acid-induced conformational variability on protein separation in reversed phase high performance liquid chromatography

Bobály

Tóth

Drahos

et al. 2014

Journal of Chromatography A

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Trends in sample preparation for classical and second generation proteomics

Cited by 166 publications

References 232 publications

Proteomic Analysis of Laser-Captured Paraffin-Embedded Tissues: A Molecular Portrait of Head and Neck Cancer Progression

Proteomic Analysis of Laser-Captured Paraffin-Embedded Tissues: A Molecular Portrait of Head and Neck Cancer Progression

Use of narrow‐range peptide IEF to improve detection of lung adenocarcinoma markers in plasma and pleural effusion

Influence of acid-induced conformational variability on protein separation in reversed phase high performance liquid chromatography

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