Investigation of the applicability of a sequential digestion protocol using trypsin and leucine aminopeptidase M for protein identification by matrix-assisted laser desorption/ionization - time of flight mass spectrometry
Bibliographic record
Abstract
An investigation of the applicability of a sequential digestion procedure involving endo- and exoprotease digestion of proteins is reported. The procedure involves the digestion of a protein sample with trypsin, yielding peptide fragments characteristic of the protein. The resulting mixture of peptide fragments is then subjected to N-terminal sequencing with leucine aminopeptidase M (LAP), with matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis of the various digestion products. Several proteins in solution, as well as gel extracted proteins were subjected to this sequential enzyme digestion procedure. The results of these experiments reveal that LAP will preferentially cleave specific peptides of the trypsin digested sample with high efficiency, while leaving other peptides undigested. Also, the length of the amino acid sequence tags that can be generated with this method is limited; the longest sequence tag generated from a single tryptic peptide was four amino acids, even though the digestion was allowed to proceed for long times. In the experiments, N-terminal digestion products were detected as early as two minutes, or as late as 90 minutes, following the addition of LAP to the sample. The method was shown to be effective for subpicomole starting quantities of protein, although with some loss in digestion efficiency at lower concentrations of starting material. This method is useful in providing additional sequence information to increase the level of confidence in protein identification, as illustrated in the identification of bacterial proteins fractionated by high-performance liquid chromatography. In some instances, this method can provide additional sequence information where post source decay and nanospray mass spectrometry failed to generate fragment ion spectra. This is illustrated in an example where the procedure was applied to a membrane protein, CD9, that had been isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Although the sequential digestion procedure requires more human intervention, it is a straightforward method and can be readily implemented.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 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) | 0.000 | 0.000 |
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 teacher head, 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".