Comparison of detergent-based sample preparation workflows for LTQ-Orbitrap analysis of the <i>Escherichia coli</i>
 proteome

Tanca, Alessandro; Biosa, Grazia; Pagnozzi, Daniela; Addis, Maria Filippa; Uzzau, Sergio

doi:10.1002/pmic.201200478

Cited by 98 publications

(93 citation statements)

References 56 publications

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“…In bacterial cells, a recent study was performed by Tanca et al, who presented a comparison of SDS/urea-FASP and ISD-RG on Escherichia coli lysates. 68 They found a slight preference of FASP over RG when assessing identification/spectrum-based parameters among around 1000 bacterial proteins. The comparison of FASP to SDC on bacterial proteomes, however, is controversially discussed in the literature, 69,70 which is likely due to individual and laboratory-specific differences in handling, instrumentation, and data analysis strategies.…”

Section: Comparison To Faspmentioning

confidence: 99%

Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells

2015

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We evaluated different in-solution and FASP-based sample preparation strategies for absolute protein quantification. Label-free quantification (LFQ) was employed to compare different sample preparation strategies in the bacterium Pseudomonas aeruginosa and human embryonic kidney cells (HEK), and organismal-specific differences in general performance and enrichment of specific protein classes were noted. The original FASP protocol globally enriched for most proteins in the bacterial sample, whereas the sodium deoxycholate in-solution strategy was more efficient with HEK cells. Although detergents were found to be highly suited for global proteome analysis, higher intensities were obtained for high-abundant nucleic acid-associated protein complexes, like the ribosome and histone proteins, using guanidine hydrochloride. Importantly, we show for the first time that the observable total proteome mass of a sample strongly depends on the sample preparation protocol, with some protocols resulting in a significant underestimation of protein mass due to incomplete protein extraction of biased protein groups. Furthermore, we demonstrate that some of the observed abundance biases can be overcome by incorporating a nuclease treatment step or, alternatively, a correction factor for complementary sample preparation approaches.

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Section: Comparison To Faspmentioning

confidence: 99%

Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells

2015

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“…Then, liquid chromatography-mass spectrometry/ mass spectrometry (LC-MS/MS) analyses of tryptic digests were performed on an LTQ Orbitrap Velos (Thermo Scientific, San Jose, CA, USA) interfaced with an UltiMate 3000 RSLCnano LC system (Dionex, Sunnyvale, CA, USA), as detailed elsewhere [31]. Peptide mixtures from gel slices were subjected to 60 min runs, comprising a 30-min gradient from 1 to 50 % eluent B (0.2 % formic acid in 95 % acetonitrile) in eluent A (0.2 % formic acid in 5 % acetonitrile).…”

Section: Western Immunoblotting and Reverse Phase Protein Arraysmentioning

confidence: 99%

Investigation of HER2 expression in canine mammary tumors by antibody-based, transcriptomic and mass spectrometry analysis: is the dog a suitable animal model for human breast cancer?

Burrai

Tanca²,

Miglio

et al. 2015

Tumor Biol.

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Canine mammary tumors (CMTs) share many features with human breast cancer (HBC), specifically concerning cancer-related pathways. Although the human epidermal growth factor receptor 2 (HER2) plays a significant role as a therapeutic and prognostic biomarker in HBC, its relevance in the pathogenesis and prognosis of CMT is still controversial. The aim of this study was to investigate HER2 expression in canine mammary hyperplasic and neoplastic tissues as well as to evaluate the specificity of the most commonly used polyclonal anti HER2 antibody by multiple molecular approaches. HER2 protein and RNA expression were determined by immunohistochemistry (IHC) and by quantitative real-time (qRT) PCR. A strong cell membrane associated with non-specific cytoplasmic staining was observed in 22% of carcinomas by IHC. Adenomas and carcinomas exhibited a significantly higher HER2 mRNA expression when compared to normal mammary glands, although no significant difference between benign and malignant tumors was noticed by qRT-PCR. The IHC results suggest a lack of specificity of the FDA-approved antibody in CMT samples as further demonstrated by Western immunoblotting (WB) and reverse phase protein arrays (RPPA). Furthemore, HER2 was not detected by mass spectrometry (MS) in a protein-expressing carcinoma at the IHC investigation. This study highlights that caution needs to be used when trying to translate from human to veterinary medicine information concerning cancer-related biomarkers and pathways. Further investigations are necessary to carefully assess the diagnostic and biological role specifically exerted by HER2 in CMTs and the use of canine mammary tumors as a model of HER2 over-expressing breast cancer.

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“…When urea is used, the temperature should not exceed 60°C as the latter results in carbamylation of proteins or peptides. Repigest is another denaturant that is becoming common in shotgun proteomics due to its compatibility with LC-MS/ MS [65,66].…”

Section: Protein Digestionmentioning

confidence: 99%

Optimized Approaches for Quantification of Drug Transporters in Tissues and Cells by MRM Proteomics

Prasad

Unadkat

2014

AAPS J

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Abstract. Drug transporter expression in tissues (in vivo) usually differs from that in cell lines used to measure transporter activity (in vitro). Therefore, quantification of transporter expression in tissues and cell lines is important to develop scaling factor for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated drug disposition. Since traditional immunoquantification methods are semiquantitative, targeted proteomics is now emerging as a superior method to quantify proteins, including membrane transporters. This superiority is derived from the selectivity, precision, accuracy, and speed of analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, LC-MS/MS proteomics has broader applicability because it does not require selective antibodies for individual proteins. There are a number of recent research and review papers that discuss the use of LC-MS/MS for transporter quantification. Here, we have compiled from the literature various elements of MRM proteomics to provide a comprehensive systematic strategy to quantify drug transporters. This review emphasizes practical aspects and challenges in surrogate peptide selection, peptide qualification, peptide synthesis and characterization, membrane protein isolation, protein digestion, sample preparation, LC-MS/MS parameter optimization, method validation, and sample analysis. In particular, bioinformatic tools used in method development and sample analysis are discussed in detail. Various pre-analytical and analytical sources of variability that should be considered during transporter quantification are highlighted. All these steps are illustrated using P-glycoprotein (Pgp) as a case example. Greater use of quantitative transporter proteomics will lead to a better understanding of the role of drug transporters in drug disposition.

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Comparison of detergent-based sample preparation workflows for LTQ-Orbitrap analysis of the Escherichia coli proteome

Cited by 98 publications

References 56 publications

Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells

Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells

Investigation of HER2 expression in canine mammary tumors by antibody-based, transcriptomic and mass spectrometry analysis: is the dog a suitable animal model for human breast cancer?

Optimized Approaches for Quantification of Drug Transporters in Tissues and Cells by MRM Proteomics

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