Nucleic Acids Research, 2006, Vol. 34, Web Server issue W261 Figure 3. A protein summary report generated by MassPective. It is a summary of the output of MODi for each identified protein, containing protein information and sequence coverage for proteins which correspond to input spectra. Also, for each interpreted spectrum, it gives a submitted mass, matched peptide sites in protein sequence, match score, a spectrum identifier and identified PTMs. Figure 4. Manual sequencing by MassPective. If a user designates an area in the spectrum, a window pops up showing all possible combinations of one amino acid mass possibly with one PTM whose mass agrees with the difference of two peaks in the area with higher intensity than the intensity value of the designated line. Another function of MassPective is to report protein summary in CSV (Comma-Seperated Values) ﬁle format (Figure 3) or save spectral image in *.bmp or *.jpeg format and print out the currently displayed spectrum. MassPective also supports manual de novo sequencing with PTMs by identifying every amino acid (possibly with a PTM) of which mass corresponds to the mass difference of any two peaks in a designated spectral segment. (Figure 4) RESULTS MODi web server has been tested by four groups: a group that uses a nano-LC/MS–MS system consisting of an Ultimate HPLC system (LC Packings) for nano-LC and a Q-TOF Ultima Global mass spectrometer (Micromass) equipped with a nano-ESI source, a group that uses a Finnigan LTQ equipped with ‘in-house’ nano-ESI source, a group that uses Finnigan LCQ equipped with ‘in-house’ nano-LC system and a group that uses Applied Biosystems 4700 Proteomics Analyzer MALDI-TOF/TOF equipped with Agilent 1100 series capillary HPLC system. Figure 5 shows how MODi interprets a spectrum with mul- tiple modiﬁcations successfully. MODi interprets the spectrum as a peptide ‘GKGGKGLGKGGAKR’ with four acetylation sites at every Lysine in the peptide. As shown in Figure 5, MODi ﬁrst ﬁnds a tag chain __GG _glg_gga__, computes the mass offsets of the gaps, which are 41.95, 41.92, 41.94 and