Protein phosphorylation analysis in archival clinical cancer samples by shotgun and targeted proteomics approaches

Gámez‐Pozo, Angelo; Sánchez‐Navarro, Iker; Calvo, Enrique; Díaz, Esther; Miguel-Martín, María; López, R.; Agulló, Teresa; Camafeita, Emilio; Espinosa, Enrique; López, Juan Antonio; Nistal, Manuel; Vara, Juan Ángel Fresno

doi:10.1039/c1mb05113j

Cited by 35 publications

(34 citation statements)

References 35 publications

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“…It has been assumed that the cross-linking of proteins prevents a proteomic analysis. Recently, it could be shown that proteins can be effectively extracted from formalin-fixed and paraffin-embedded samples and that the proteins and phosphorylations are quantitatively preserved compared with fresh frozen tissues (56,61,62).…”

Section: Resultsmentioning

confidence: 99%

Phosphosignature Predicts Dasatinib Response in Non-small Cell Lung Cancer

Klammer

Kaminski

Zedler

et al. 2012

Molecular & Cellular Proteomics

102

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Targeted drugs are less toxic than traditional chemotherapeutic therapies; however, the proportion of patients that benefit from these drugs is often smaller. A marker that confidently predicts patient response to a specific therapy would allow an individual therapy selection most likely to benefit the patient. Here, we used quantitative mass spectrometry to globally profile the basal phosphoproteome of a panel of non-small cell lung cancer cell lines. The effect of the kinase inhibitor dasatinib on cellular growth was tested against the same panel. From the phosphoproteome profiles, we identified 58 phosphorylation sites, which consistently differ between sensitive and resistant cell lines. Many of the corresponding proteins are involved in cell adhesion and cytoskeleton organization. We showed that a signature of only 12 phosphorylation sites is sufficient to accurately predict dasatinib sensitivity. The introduction of targeted drugs for treating cancer is a major biomedical achievement of the past decade (1, 2). Because these drugs selectively block molecular pathways that are typically overactivated in tumor cells, they are more precise and less toxic than traditional chemotherapeutics. However, although many cancer patients benefit from a specific targeted therapy, many others do not. Therefore, predictive molecular markers are needed to confidently predict patient response to a specific therapy. Such markers would facilitate therapy personalization, where the selected therapy is based on the molecular profile of the patient.Predictive tests currently used in the clinic are frequently based on one particular marker that is often linked to the drug target. A well known example for a predictive test is assessing HER2/neu overexpression using immunohistochemistry or fluorescent in situ hybridization to predict the response to therapy with trastuzumab (Herceptinா; Roche) (3, 4). However, in some cases the expression or mutational status of the target or other singleton markers might not be sufficient to predict a therapeutic response. Recently, several studies tried to identify molecular signatures comprising multiple markers for response predictions, usually based on gene expression profiling (5, 6). To our knowledge, no study successfully identified a signature from global phosphoproteomic profiles so far.Recent advances in mass spectrometry, methods for enriching phosphorylated proteins or peptides, and computer algorithms for analyzing proteomics data have enabled the application of mass spectrometry-based proteomics to monitor phosphorylation events in a global and unbiased manner. These methods have become sufficiently sensitive and robust to localize and quantify the phosphorylation sites within a peptide sequence (7-9). Phosphorylation events are important in signal transduction, where signals caused by external stimuli are transmitted from the cell membrane to the nucleus. Aberrations in these signal transduction pathways are particularly important for understanding the mechanisms of certain diseases,...

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Section: Resultsmentioning

confidence: 99%

Phosphosignature Predicts Dasatinib Response in Non-small Cell Lung Cancer

Klammer

Kaminski

Zedler

et al. 2012

Molecular & Cellular Proteomics

102

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“…tides in the assay and it is technically possible to retrieve phosphopeptides from FFPE tissues (41). However, the question remains whether this would be a reliable approach for FFPE tissues.…”

Section: Targeted Mass Spectrometry In Formalin-fixed Tissuesmentioning

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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“…Briefly, FFPE sections were deparaffinized in xylene and washed twice with absolute ethanol. Protein extracts from FFPE samples were prepared in 2% SDS buffer using a protocol based on heat-induced antigen retrieval (6). Protein concentration was determined using the MicroBCA Protein Assay Kit (Pierce-Thermo Scientific).…”

Section: Total Protein Preparationmentioning

confidence: 99%

“…Fresh or fresh-frozen specimens are ideal for molecular profiling but rarely available in the clinic. In recent years, we and others have demonstrated that formalinfixed, paraffin-embedded (FFPE) tissue can be readily analyzable by MS-based technologies (6,7). On the other hand, previous studies demonstrate the possibility of performing experiments to analyze the expression of miRNAs in FFPE tissues (8).…”

Section: Introductionmentioning

confidence: 99%

Combined Label-Free Quantitative Proteomics and microRNA Expression Analysis of Breast Cancer Unravel Molecular Differences with Clinical Implications

et al. 2015

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Better knowledge of the biology of breast cancer has allowed the use of new targeted therapies, leading to improved outcome. High-throughput technologies allow deepening into the molecular architecture of breast cancer, integrating different levels of information, which is important if it helps in making clinical decisions. microRNA (miRNA) and protein expression profiles were obtained from 71 estrogen receptor-positive (ER þ ) and 25 triple-negative breast cancer (TNBC) samples. RNA and proteins obtained from formalin-fixed, paraffin-embedded tumors were analyzed by RT-qPCR and LC/MS-MS, respectively. We applied probabilistic graphical models representing complex biologic systems as networks, confirming that ER þ and TNBC subtypes are distinct biologic entities. The integration of miRNA and protein expression data unravels molecular processes that can be related to differences in the genesis and clinical evolution of these types of breast cancer. Our results confirm that TNBC has a unique metabolic profile that may be exploited for therapeutic intervention. Cancer Res; 75(11); 2243-53. Ó2015 AACR.

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Protein phosphorylation analysis in archival clinical cancer samples by shotgun and targeted proteomics approaches

Cited by 35 publications

References 35 publications

Phosphosignature Predicts Dasatinib Response in Non-small Cell Lung Cancer

Phosphosignature Predicts Dasatinib Response in Non-small Cell Lung Cancer

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

Combined Label-Free Quantitative Proteomics and microRNA Expression Analysis of Breast Cancer Unravel Molecular Differences with Clinical Implications

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