Despite superior analytical features, mass spectrometry proteomics remains seldom used for the basic investigation and clinical treatment of human disease. This need is particularly pressing for childhood diseases that can be rare in incidence and variable in presentation. Modern mass spectrometry enables detailed functional characterization of the pathogenic biochemical processes, as achieved by accurate and comprehensive quantification of proteins and their regulatory chemical modifications. Here, we describe how high-accuracy mass spectrometry in combination with high-resolution chromatographic separations can be leveraged to meet these analytical requirements in a mechanism-focused manner. We review the quantification methods capable of producing accurate measurements of protein abundance and post-translational modification stoichiometries. We then discuss how experimental design and chromatographic resolution can be leveraged to achieve comprehensive functional characterization of biochemical processes in complex biological proteomes. Finally, we describe current approaches for quantitative analysis of a common functional protein modification: reversible phosphorylation. In all, current instrumentation and methods of high-resolution chromatography and mass spectrometry proteomics are poised for immediate translation into improved diagnostic and therapeutic strategies for pediatric and adult diseases.