Towards an optimized workflow for middle-down proteomics

Detailed description of an optimally tuned workflow for high throughput middle-down proteomics with the currently available state-of-the-art instrumentation. In this work we describe and critically evaluate the limits of an optimized workflow for the detection of middle-range peptides. First, the work compares the yield of desired longer peptides both by enzymatic and acidic digestion protocols. Second, it uses optimized conditions for sample clean-up and multidimensional LC. Third, we fine-tuned critical MS parameters for improving the detection of middle-range peptides where we comprehensively evaluated ETD, HCD and EThcD fragmentation schemes on all the digests obtained in order to choose the best sequencing method for distinct populations of peptides and with special attention paid to longer peptides.