A better understanding of the scan-to-scan signal intensity variation can lead to more sophisticated algorithms for database searching and de novo peptide sequencing using single scan mass spectra. In this study, we systematically studied the variation in relative intensity of m/z values in the single scan product ion mass spectra (MS2) derived from five representative precursor ions (MS1) collected using an LTQ linear ion trap under constant flow direct infusion conditions with peptide concentrations held constant. We applied a matching algorithm based on a pair hidden Markov model to align the peaks from each scan belonging to the same m/z value prior to assessing the signal intensity variation. The most significant single contributor to scan-to-scan signal intensity variation for high abundance ions was centroider error. Our study also showed that the variation in signal intensity is higher than what would be expected if the ion statistics derived from the dual geometry electron multiplier detector followed a Poisson distribution.