AbstractMass spectrometry-based methods have been widely applied – often as the sole method – to detect mutations in human cancer specimens. We applied this approach to 52 childhood soft tissue sarcoma specimens in an attempt to identify potentially actionable mutations. This analysis revealed that 25% of the specimens harbored high-confidence calls for mutated alleles, including a mutation encoding FLT3^I836M that was called in four cases. Given the surprisingly high frequency and unusual nature of some of the mutant alleles, we carried out ultra-deep next generation sequencing to confirm them. We confirmed only three mutations, which encoded NRAS^A18T, JAK3^V722I and MET^R970C in three specimens. Beyond highlighting those mutations, our findings demonstrate potential pitfalls of primarily utilizing a mass spectrometry-based approach to broadly screen for DNA sequence variants in archived, clinical-grade tumor specimens. Duplicate mass spectrometric analyses and confirmatory next generation sequencing can help diminish false positive calls, but this does not ameliorate potential false negatives due in part to evaluating a limited panel of sequence variants.