Quantification Methods Compared

Label-Free Quantification (LFQ)

LFQ compares peptide intensities (MS1 or MS2) across runs without isotopic labels. It is the most cost-effective and flexible approach, compatible with any sample type. Normalisation is critical; we use median ratios, LOESS, or variance-stabilising normalisation depending on the data.

Pros: Unlimited comparisons, no labelling cost, simple workflow. Cons: Higher missing values, run-to-run variability.

TMT (Tandem Mass Tags)

TMT labels peptides from up to 18 samples with isobaric tags (TMTpro 32-plex reagents now allow up to 35), enabling multiplexed quantification in a single LC-MS run. Reporter ions in MS2 provide relative quantification. TMT reduces missing values and is ideal for time-course or multi-condition experiments.

Pros: Minimal missing values, high throughput, ratio compression manageable with MS3 or SPS-MS3. Cons: Higher reagent cost, requires higher-resolution instrumentation for MS3.

Targeted Quantification (PRM/SRM)

Parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) provide the highest specificity and sensitivity for quantifying known peptides. Ideal for validation studies, clinical assays, and measuring low-abundance targets.