Comparison of label-free and label-based strategies for proteome analysis of hepatoma cell lines

Megger, Dominik A.; Pott, Leona L.; Ahrens, Maike; Padden, Juliet; Bracht, Thilo; Kuhlmann, Katja; Eisenacher, Martin; Meyer, Helmut E.; Sitek, Barbara

doi:10.1016/j.bbapap.2013.07.017

Cited by 106 publications

(98 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…11 Four replicates of each cell line were prepared. Confluent monolayers of approximately 5 × 10 6 cells were washed with PBS and cells were carefully detached with enzyme-free cell dissociation buffer (Thermo Scientific, Bremen, Deutschland) and centrifuged for 5 min at 800g.…”

Section: Cell Culture and Labeling Reactionsmentioning

confidence: 99%

“…11 Here, 100 μg of reduced and alkylated protein sample was used for labeling reaction. After in-gel digestion of the labeled samples, equal peptide amounts were combined according to the scheme shown in Table 1.…”

Section: Cell Culture and Labeling Reactionsmentioning

confidence: 99%

“…After reduction and alkylation, the samples were subjected to in-gel digestion as described earlier. 11 The generated peptides were extracted and quantified by amino acid analysis. Equal amounts of the peptide extracts from six differentially labeled samples, corresponding to two replicates of each cell line per sample set, were combined.…”

Section: Cell Culture and Labeling Reactionsmentioning

confidence: 99%

“…11 Here, 13 μg of tryptic peptides derived from in-gel digestion of reduced and alkylated protein samples were labeled using amine-reactive TMT reagent. Differentially labeled samples (1 μg of each condition) were mixed according the mixing scheme shown in Table 1.…”

Section: Cell Culture and Labeling Reactionsmentioning

confidence: 99%

“…9 Usually, these tags are incorporated via the reaction of primary amines of peptide N-termini and lysine residues with a reactive NHS-ester group of the tagging reagent. Whereas labeling reactions at the levels of isolated proteins or peptides derived from a proteolytic digestion are well established and applied to tackle various research questions, [10][11][12][13][14][15][16][17] the introduction of tandem mass tags at the level of intact cells has not been reported yet, even though labeling of whole cells with amine-reactive reagents is widely used and described. In the context of gel-based proteome analysis, for example, Mayrhofer et al performed labeling of intact cells with amine-reactive CyDye DIGE fluorophors enabling for a specific and more sensitive quantification of surface proteins.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

NHS-based Tandem Mass Tagging of Proteins at the Level of Whole Cells: A Critical Evaluation in Comparison to Conventional TMT-Labeling Approaches for Quantitative Proteome Analysis

et al. 2017

Self Cite

View full text Add to dashboard Cite

Tandem mass tags (TMT) are usually introduced at the levels of isolated proteins or peptides. Here, for the first time, we report the labeling of whole cells and a critical evaluation of its performance in comparison to conventional labeling approaches. The obtained results indicated that TMT protein labeling using intact cells is generally possible, if it is coupled to a subsequent enrichment using anti-TMT antibody. The quantitative results were similar to those obtained after labeling of isolated proteins and both were found to be slightly complementary to peptide labeling. Furthermore, when using NHS-based TMT, no specificity towards cell surface proteins was observed in the case of cell labeling. In summary, the conducted study revealed first evidence for the general possibility of TMT cell labeling and highlighted limitations of NHS-based labeling reagents. Future studies should therefore focus on the synthesis and investigation of membrane impermeable TMTs to increase specificity towards cell surface proteins.

show abstract

Section: Cell Culture and Labeling Reactionsmentioning

confidence: 99%

Section: Cell Culture and Labeling Reactionsmentioning

confidence: 99%

Section: Cell Culture and Labeling Reactionsmentioning

confidence: 99%

Section: Cell Culture and Labeling Reactionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

NHS-based Tandem Mass Tagging of Proteins at the Level of Whole Cells: A Critical Evaluation in Comparison to Conventional TMT-Labeling Approaches for Quantitative Proteome Analysis

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

Quantitative Proteomics Techniques in Biomarker Discovery

Bracht

Megger

Naboulsi

et al. 2015

Biomarker Validation

View full text Add to dashboard Cite

Enhanced reproducibility of the human gel‐based tear proteome maps in the presence of di‐(2‐hydroxyethyl) disulfide

Saraygord‐Afshari

Naderi-Manesh

Naderi

2014

Biotech and App Biochem

View full text Add to dashboard Cite

Patterns obtained in two-dimensional gel electrophoresis (2-DE) in the previously published articles suggest a varying number of proteins. To seek the cause of this variation, we investigated the effect of reduction power on the overall tear proteome maps. To this end, the buffers of two reducing agents, dithiothreitol (DTT) at nine different concentrations and di-(2-hydroxyethyl) disulfide (HED), were examined. The assay showed that HED clearly improved 2-DE resolution, increased the number of detectable protein spots, and offered well-resolved chain regions in comparison with those treated with DTT. Furthermore, this study introduced increasing the reduction power as a remedy to increase the reproducibility of two-dimensional human tear proteome maps. In addition, the results of our assessment showed that improved reduction efficiency was accompanied by increased procedure reproducibility from 42% to 89%.

show abstract

Comparison of label-free and label-based strategies for proteome analysis of hepatoma cell lines

Cited by 106 publications

References 30 publications

NHS-based Tandem Mass Tagging of Proteins at the Level of Whole Cells: A Critical Evaluation in Comparison to Conventional TMT-Labeling Approaches for Quantitative Proteome Analysis

NHS-based Tandem Mass Tagging of Proteins at the Level of Whole Cells: A Critical Evaluation in Comparison to Conventional TMT-Labeling Approaches for Quantitative Proteome Analysis

Quantitative Proteomics Techniques in Biomarker Discovery

Enhanced reproducibility of the human gel‐based tear proteome maps in the presence of di‐(2‐hydroxyethyl) disulfide

Contact Info

Product

Resources

About