Proteomics studies the proteins present in a biological system, including their abundance, modifications and interactions. A useful experiment starts with a specific question: identify an unknown protein, compare groups, measure a small set of targets, or investigate a modified form. That question determines the sample handling, measurement and evidence needed.
This guide is for students and researchers discussing a study with a proteomics facility. It explains choices and report checks; the facility's validated procedures govern sample preparation and instrument operation. EMBL-EBI's proteomics introduction places abundance, modification and interaction studies within the broader field.
Choose the measurement around the question
| Question | Possible approach | What to establish |
|---|---|---|
| Which proteins are present? | Discovery mass spectrometry, often liquid chromatography with tandem mass spectrometry (LC-MS/MS). | Sample coverage, peptide evidence and identification error control. |
| Which proteins differ between groups? | Quantitative discovery proteomics with a suitable label-free or labeling strategy. | Independent biological samples, balanced batches and a prespecified comparison. |
| How much of a selected target is present? | Targeted MS or a validated affinity assay. | Specificity, calibration, working range and the sample matrix. |
| Is a protein modified or interacting? | A method designed for the modification or interaction, sometimes with enrichment. | Site confidence, controls and whether enrichment changes the population measured. |
MALDI describes a way of creating ions using a matrix and laser. LC describes a separation step; MS/MS adds fragmentation evidence. These terms describe different parts of a measurement system. An instrument or consumable alone cannot define the biological question. For a proposed workflow, ask the facility to describe the path from your actual specimen to the reported result.
Follow the evidence from sample to protein
In a common bottom-up workflow, proteins are broken into peptides, separated and measured. Software compares fragmentation patterns with candidate peptide sequences, then assembles peptide evidence into protein identifications. Shared peptides may fit several related proteins, so a reported protein group can be more defensible than a single named isoform. EMBL-EBI's identification training covers spectrum matching, validation, decoys and false discovery rate.
Ask which error rate applies to spectrum matches, peptides and proteins. A stated 1% false discovery rate is an estimate for a selected set under the analysis assumptions; it does not assign every individual identification a 99% probability of being correct. Identification error control also differs from correcting thousands of abundance comparisons for multiple testing.
Record the sequence database and release, software and version, search settings, modification choices and filtering thresholds. Those details let another analyst understand why a protein appeared in the list.
Design comparisons before collecting samples
Define the biological unit first: independent people, animals, cultures or specimens. Repeated instrument injections help characterize measurement repeatability; they do not create additional independent biological samples. Allocate groups across preparation and acquisition batches so that treatment and processing date can be separated.
Agree on sample acceptance criteria with the facility, including material, amount, collection conditions and known contaminants. Set a plan for pooled quality-control samples, blanks, run order and failed runs. Decide how missing measurements will be examined before looking for a desirable difference. Missingness can reflect low abundance, sampling or a technical problem; automatically turning every missing value into zero can manufacture a contrast.
The EMBL-EBI course materials connect experimental design, quantification and downstream analysis. Use them to prepare questions for the scientist who will analyze your experiment.
Read an abundance difference in context
If the finding will support an important decision, plan confirmation in independent material using an appropriate complementary measurement. Agreement should concern the same target, specimen type and biological contrast. A different assay may recognize a different protein form.
What to request with a report
- The sample sheet, biological groups, run order and exclusions with reasons.
- Quality-control plots and a record of batch or instrument problems.
- Protein and peptide tables, identifier mappings and filtering rules.
- The normalization, missing-value and statistical methods, with effect sizes and uncertainty.
- Raw-data access or repository identifiers and the analysis settings needed for reuse.
Use PRIDE to locate deposited mass-spectrometry datasets and supporting files. Confirm that the accession belongs to the reported experiment before reusing it. A useful final summary states what was measured, which comparison was made, what changed, and what still needs validation.