Shotgun proteomics of archival triple‐negative breast cancer samples

Gámez‐Pozo, Angelo; Ferrer, Nuria Ibarz; Ciruelos, Eva; López-Vacas, Rocío; Martínez, Fernando García; Espinosa, Enrique; Vara, Juan Ángel Fresno

doi:10.1002/prca.201200048

Cited by 33 publications

(30 citation statements)

References 56 publications

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“…Specifically, the DT workflow entails an initial tissue homogenization, which can be performed using different buffers, such as the commercial Liquid Tissue [16], as well as mixtures of ammonium bicarbonate (ABC) and acetonitrile (ACN) [17,18] or ABC and trifluoroethanol [19,20], followed by direct trypsin digestion of the tissue homogenate. The ISD workflow is instead based on a preliminary protein extraction step, comprising tissue lysis in a detergent-based buffer (usually containing SDS) and collection of soluble proteins after centrifugation, succeeded by detergent depletion (by means of dilution with ABC [21,22], dialysis [23], spin columns [24] or protein precipitation with various protocols [25-28]) and in solution digestion of the protein extract. The FASP protocol, applied with remarkable results to FFPE samples by Wiśniewski and colleagues [29-32] and recently employed with modifications by other research groups [33,34], shares the initial protein extraction step with the ISD workflow but differs in that detergent removal and protein digestion are both performed on a molecular weight cut-off centrifugal filter [35,36], instead of in solution.…”

Section: Introductionmentioning

confidence: 99%

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

et al. 2014

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BackgroundThe growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking.Experimental designDT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity.ResultsDT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility.ConclusionsThese results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.

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Section: Introductionmentioning

confidence: 99%

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

et al. 2014

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“…We have previously demonstrated that our workflow is high reproducible (10). One hundred and two FFPE samples yielded enough protein to perform the MS analyses (typically, more than 0.5 mg of total protein was recovered from each sample).…”

Section: Protein Extraction and Ms Analysismentioning

confidence: 99%

“…Proteins were extracted from FFPE samples as previously described (10). Briefly, FFPE sections were deparaffinized in xylene and washed twice with absolute ethanol.…”

Section: Total Protein Preparationmentioning

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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“…In addition, protein detection is probably also more reflective of the tumor microenvironment. Several proteomic studies have been conducted on TNBC (3)(4)(5), but no proteomic study was conducted on large cohorts including the clinical outcome of the patients, except a recent comparative proteome analysis that identified a 11-protein signature for aggressive TNBC in a large cohort of 93 microdissected tumors (6). Although microdissection was necessary to elucidate the contribution of TNBC cells, it did not reflect the tumor with its microenvironment that is increasingly described as fundamental to explain the tumor outcome.…”

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confidence: 99%

Prediction of Recurrence and Survival for Triple-Negative Breast Cancer (TNBC) by a Protein Signature in Tissue Samples

Campone

Valo

Jézéquel

et al. 2015

Molecular & Cellular Proteomics

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To date, there is no available targeted therapy for patients who are diagnosed with triple-negative breast cancers (TNBC). The aim of this study was to identify a new specific target for specific treatments. Frozen primary tumors were collected from 83 adjuvant therapynaive TNBC patients. These samples were used for global proteome profiling by iTRAQ-OFFGEL-LC-MS/MS approach in two series: a training cohort (n ‫؍‬ 42) and a test set (n ‫؍‬ 41). Patients who remains free of local or distant metastasis for a minimum of 5 years after surgery were classified in the no-relapse group; the others were in the relapse group. OPLS and Kaplan-Meier analyses were performed to select candidate markers, which were validated by immunohistochemistry. Three proteins were identified in the training set and validated in the test set by Kaplan-Meier method and immunohistochemistry (IHC): TrpRS as a good prognostic markers and DP and TSP1 as bad prognostic markers. We propose the establishment of an IHC test to calculate the score of TrpRS, DP, and TSP1 in TNBC tumors to evaluate the degree of aggressiveness of the tumors. Finally, we propose that DP and TSP1 could provide therapeutic targets for specific treatments. Molecular & Cellular

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Shotgun proteomics of archival triple‐negative breast cancer samples

Abstract: Our results open up new avenues for the use of proteomics technologies in clinically relevant studies using archival samples. Shotgun LC-MS/MS studies could serve to discover new biomarkers and may provide clues to the genesis of TNBC and underlying molecular alterations.

Cited by 33 publications

References 56 publications

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

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

Prediction of Recurrence and Survival for Triple-Negative Breast Cancer (TNBC) by a Protein Signature in Tissue Samples

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