Middle-down proteomics has emerged as the method of choice to study 
combinatorial histone post translational modifications (PTMs). In the common 
bottom-up workflow, histones are digested into relatively short peptides (4-20 
aa), separated using reversed-phase chromatography and analyzed using typical 
proteomics methods in mass spectrometry. In middle-down, histones are cleaved 
into longer polypeptides (50-60 aa) mostly corresponding to their N-terminal 
tails, resolved using weak cation exchange-hydrophilic interaction liquid 
chromatography (WCX-HILIC) and analyzed with less conventional mass 
spectrometry, i.e. using Electron Transfer Dissociation (ETD) for analyte 
fragmentation. Middle-down is not nearly as utilized as bottom-up for PTM 
analysis, partially due to its limited reproducibility and robustness. This has 
also limited the establishment of rigorous benchmarks to discriminate good vs 
poor quality experiments. Here, we describe critical aspects of the middle-down 
workflow to assist the user in evaluating the presence of biased and misleading 
results. Specifically, we tested the use of porous graphitic carbon (PGC) during 
the desalting step, demonstrating that desalting using only C18 material leads 
to sample loss. We also tested different salts in the WCX-HILIC buffers for 
their effect on retention, selectivity, and reproducibility of analysis of 
variants of histone tail fragments, in particular replacing ammonium ion with 
ethylenediammonium ion in buffer A. These substitutions had marked effects on 
selectivity and retention. Our results provide a streamlined way to evaluate 
middle-down performance to identify and quantify combinatorial histone PTMs.