Improved Detection of Quantitative Differences Using a Combination of Spectral Counting and MS/MS Total Ion Current

Label-free quantitative strategies are commonly used in shotgun proteomics to detect differences in protein abundance between biological sample groups. Here, we have employed a combination of two such approaches, spectral counting (SpC) and average MS/MS total ion current (MS² TIC), for the analysis of rat kidney mitochondria in response to metabolic acidosis. In total, 49 proteins were observed to be significantly altered in response to metabolic acidosis (p-value < 0.05). Of these, 32 proteins were uniquely observed as significantly different by SpC, 14 by MS² TIC, and only 3 by both approaches. Western blot analysis was performed on a subset of these proteins to validate the observed abundance differences. This study illustrates the utility and ease of combining these two label-free quantitative approaches to increase the number of detected protein abundance differences in the shotgun analysis of complex biological samples.