A proteomic analysis combining peptide de novo sequencing and BLAST analysis was used to identify novel proteins involved in copper tolerance in the marine alga Scytosiphon gracilis (Phaeophyceae). Algal material was cultivated in seawater without copper (control) or supplemented with 100 mu g L(-1) for 4 days, and protein extracts were separated by two-dimensional gel electrophoresis (2-DE). From the proteins obtained in the copper treatment, 25 over-expressed, 5 under-expressed and 5 proteins with no changes as compared with the control, were selected for sequencing. Tryptic-peptides obtained from 35 spots were analyzed by capillary liquid chromatography and tandem mass spectroscopy (capLC/MS/MS). and protein identity was determined by BLASTP. We identified 19 over-expressed proteins, including a chloroplast peroxiredoxin, a cytosolic phosphomannomutase, a cytosolic glyceraldehyde-3-phosphate dehydrogenase, 3 ABC transporters, a chaperonine, a subunit of the proteasome and a tRNA synthase, among others. The possible involvement of these over-expressed proteins in buffering oxidative stress and avoiding metal uptake in S. gracilis exposed to copper excess is discussed taking into consideration the information available for other plant models. (C) 2009 Elsevier B.V. All rights reserved.