To the Editor: Large sequence-based datasets are often scanned for conserved sequence patterns to extract useful biological information1. Sequence logos2 have been developed to visualize conserved patterns in oligonucleotide and protein sequences and rely on enabled us to combine the advantages of in-gel and in-solution digestion workflows. It has been commonly held that SDS, once introduced into the sample, will make subsequent mass spectrometric analysis impossible. Manza et al. 3 explicitly state in their paper that they could not completely remove SDS and that its presence reduced the number of identified BSA peptides. Indeed, after multidimensional separation Manza et al. 3 identified 75 soluble cytosolic and 142 nuclear proteins. In contrast, our FASP approach allowed us to identify more than 7,000 proteins, about one-third of which were membrane or membrane-associated proteins2. In FASP, SDS is dissociated from proteins using urea. This presumably sequesters them into small micelles, which can pass through the filter pores, thus separating protein and detergent. The method of Manza et al. 3 does not use such a step and is therefore not effective at removing SDS or other detergents. FASP achieves essentially complete protein unfolding during the whole process of detergent removal, which allows use of largemolecular-weight cut-off filters without a loss of small proteins. In contrast, Manza et al. 3 reported that it was necessary to limit filter size to the 3–5 kDa range. The ability of FASP to work with larger pore filters substantially reduces sample preparation time. Liebler and Ham1 also state that the method is not “universal” because it disproportionately loses protein at low sample loads. We did not specifically develop the FASP protocol for high-sensitivity work. However, we demonstrated identification of 1,700 proteins from HeLa cell material corresponding to only 1,250 cells (750 ng total protein) 2. We have now tested FASP with tenfold lower amounts and did not observe a disproportionate reduction in peptide ion current, peptide or protein identifications (Supplementary Note). Current commercial spin filters are not optimized for FASP, and they are not optimal for working with very small protein amounts (< 100 ng). Miniaturization of the filter units should reduce proteins losses proportionally. In any case, in describing FASP method as “universal,” we were specifically referring to its ability to represent the proteome in an unbiased way, which we demonstrated by comparison to the transcriptome.