As expected, the identification of tubulin became more tenuous with increased stringency on accuracy. However, employing some of the tools suggested by the spectrum averaging experiments for the standard digests provided enhanced identification of tubulin. The standard peptide experiments demonstrated a standard deviation between 0.010�C0.014 amu. In the Tubulin IP experiment, 5 of the 12 peptides identified, exhibit deviations above this range. Two of these peptides were identified by LC-MS/MS as derived from tubulin. Four deviations are minor, but with restricting the peptides for a second round of analysis to those identified to tubulin in the initial fingerprinting analysis and removing the two peptides with high standard deviations, the identification of tubulin was enhanced. Such an approach improves the confidence of a correct identification, but further experiments using alternative techniques are necessary for confirmation. The averaging approach used in this study was tested up to 2465.1983 amu because of our confirmation of correct identification of peptides by MS/MS. The tuning and calibration for our 4800 MALDI-TOF/TOF system is optimized for this lower mass range because of high performance in the lower masses and problematic and unreliable peptide fragmentation above approximately 3500�C4000 amu. However, the reflectron system on this instrument is capable of higher mass analyses for mass measurements of parent masses, but not for fragment masses. We tested the higher mass reflectron system on our instrument using Insulin with the acknowledged limitations that the reflectron system was not tuned for this higher mass. As a consequence, the observed data has lower sensitivity, the resolution is unable to A 350619 hydrochloride define monoisotopic peaks, and observed masses are consistent with 3-Deazaneplanocin A hydrochloride average mass rather than monoisotopic mass. Despite these issues, we observed exactly the same type of variability as observed in the lower mass range with a majority of single mass observations inferior to the average of the population of mass observations. We fully expect that proper tuning and calibration of the reflectron system of the 4800 MALDI-TOF/TOF for higher masses such as insulin would provide similar advantages as observed in the properly tuned and calibrated lower mass region. However, the capabilities of this instrument to provide high accuracy reflectron data across a wide mass range from <1000 to >5000 amu will require further experiments. These data demonstrate that, given that similar analog-to-digital detection systems are utilized in all current MALDI-TOF instruments and that the same detection systems are used in both reflectron and linear modes, the averaging methods described in this article should be applicable across all mass ranges and modes. Obviously, reflectron data will be limited to lower mass range than linear data, but exhibit much higher accuracy as long as the reflectron system is tuned and calibrated for the appropriate mass ranges under study.