Optimization of protein identifications through the use of different chromatographic approaches and bioinformatic pipelines
Bibliographic record
Abstract
RATIONALE: Selection of proteomic workflows for a given project can be a daunting task. This research provides a guide outlining the impact on protein identification of different steps such as chromatographic separation, data acquisition strategies, and bioinformatic pipelines. The data presented here will help experts and nonexpert proteomic users to increase proteome coverage and peptide identification. METHODS: HeLa protein digests were analyzed through different C18 chromatographic columns (15 and 50 cm in length), using top 12 data-dependent acquisition (DDA), top 20 DDA, and data-independent acquisition (DIA) with a nanospray source in positive mode in a Thermo Q Exactive instrument. The raw data were analyzed using different search engines, rescoring approaches, and multi-engine searches. The results were analyzed in the context of peptide and protein identifications, precursor properties, and computation requirements to understand the differences between methods. RESULTS: Our results showed that higher column lengths and top N DDA approaches were able to significantly increase protein identifications. The use of multiple search engines yielded limited gains, whereas the use of rescoring methods clearly outperformed other strategies. Finally, DIA approaches, although successful at generating new identifications, had a limited performance influenced by the previous collection of DDA data, which could prohibitively increase instrument time. Nonetheless, the use of library-free methods showed promising results. CONCLUSIONS: Our results highlight the impact of different experimental approaches on proteome coverage. Changes in chromatographic columns, data acquisition, or bioinformatic analysis can significantly increase the number of protein identifications (>400%). Thus, this research provides a reference upon which to build a successful proteomic workflow with different considerations at every step.
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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 | 0.008 | 0.009 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.002 | 0.002 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.003 | 0.003 |
| Open science | 0.002 | 0.002 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.002 |
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".