Step	Description	Time requirements
	Starting material: SILAC heavy and light labelled HeLa cells (1 x15 cm dish each, 50% confluent, ie 2 x 10 million cells).
1	Mechanical cell lysis, and differential centrifugation subcellular fractionation → the actual ‘Fractionation Profiling’	4 hr
2	Protein assay of fractions, overnight tryptic digest, peptide clean-up	4 hr hands-on, + overnight digestion
3	Mass spectrometry analysis (Thermo Q-Exactive HF)	20 hr (fast protocol)
4	MaxQuant data processing (free software), data filtering	< 24 hr (processor with 8 cores e.g. intel i7)
5	Visualization of maps by PCA, check clustering (using eg SIMCA software (free demo), or Perseus software (free)	1 hr
6	Prediction of protein subcellular localization by SVM classification (Perseus, free software)	1 hr
→From cells to map in 3 days