Differential proteomics of metastatic and non-metastatic lung carcinoma cell lines
Bibliographic record
Abstract
4548 Two lung carcinoma cell lines, CL1-0 and CL1-5 [Chu, Y. W. et al. Am. J. Respir.Cell. Mol. Biol., 17: 353-360, 1997] are different in their invasive and metastatic potential. Both cell lines form tumors when injected to mice, but only CL1-5 cells lead to tumors capable of metastasizing to lungs. For the purpose of this work we used subcellular fractions obtained from CL1-0 and CL1-5 cells. Proteins in the cytosol were labeled with two different fluorescent dyes. We chose Alexa Fluor 555 and Alexa Fluor 647 C 2 maleimide derivatives (Invitrogen) for their stability, similar molecular weight and affordability. The cytosol proteins were labeled with the excess of the dye - each sample with a different dye. Equal amounts of labeled CL1-0 and CL1-5 cytosols were mixed and proteins resolved by 2D-gel electrophoresis. The 2D-gels were scanned (Typhoon, GE Healthcare) and protein spots belonging to either CL1-0, or to CL1-5 were identified. In addition to the fluorescence overlay image, proteins were visualized by silver staining, extracted from the gel and subjected to in-gel tryptic digestion and LC-MS/MS analysis. To verify reproducibility of labeling and separation by 2-D PAGE we inversed the labeling and repeated the proteomic analysis. Due to a higher molecular weight of Alexa Fluor 647 C 2 maleimide (approx.50 Da), saturation labeling of low-molecular-weight proteins resulted in a small, but easy to recognize shift of the spots on the overlay image. Importantly, isoelectric point was the same for proteins labeled either with Alexa Fluor 555, or Alexa Fluor 647. In both experiments we observed the same unique spots. Unique protein spots were observed mainly in CL1-0 and absent, or decreased in CL1-5 cytosol samples, i.e., down-regulated in more metastatic carcinoma cell line. Among the proteins down-regulated in CL1-5 cells were annexin I (ANX1), heat shock protein 27 (HSP 27) and down-regulated ovarian cancer 1 (DOC1), all three already established cancer markers. Somewhat unexpected was identification of the biliverdin IX beta reductase (AAB29537), a protein shown to have an antiapoptotic activity. Western blot analysis confirmed a high level of HSP27 and ANX1 in cytosols from CL1-0 cells, and a much lower signal for these proteins was observed in CL1-5 cells. In overall, we have demonstrated that Alexa Fluor dyes can be used successfully in the differential gel electrophoresis and subsequent proteomic identification of the cancer protein markers.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".