Ask a protein pro - Tips for preparing high-quality samples for mass spectrometry analysis

Learn the key techniques encompassing sample preparation for mass spectrometry, including lysate preparation, protein enrichment, protein digestion, and sample cleanup. Proper sample prep is critical because quality and reproducibility directly impact mass spec results.

Use the Thermo Scientific easy pep MS sample prep workflow to efficiently process cultured mammalian cells, tissues, and biofluids. The kit provides pre-formulated reagents that reduce processing time to under 3 hours and support samples ranging from 1 µg to 2 mg.

Deplete plasma samples of high-abundance proteins such as albumin and immunoglobulins that obscure low-abundance protein detection. Extend the digestion period to 2-3 hours to achieve less than 10% missed cleavages.

Homogenize tissue samples in lysis buffer, allowing longer incubation time for dense tissues like heart tissue. Centrifuge following homogenization to remove insoluble debris.

Assess FFPE block quality using H&E staining and use at least six micron thickness curls (minimum three curls). Homogenize samples in easy pep lysis buffer after paraffin removal and rehydration.

Use protein and peptide assays to normalize sample amounts before TMT reagent labeling. This ensures equal amounts of peptides are labeled across different samples for accurate quantification.

Apply TMT or TMT Pro reagents to label up to 18 different peptide samples. Equilibrate TMT vial to room temperature before opening to prevent moisture condensation, and ensure all amine-containing buffers are removed to maintain reactivity with lysine residues.