Despite no obvious trends in the types of proteins identified through FASP II vs acetone precipitation, Table 4 demonstrates relatively low overlap in the identified proteins (69 & 77%) and pep- tides (42 & 41%) for the low and high MW GELFrEE fractions. The acetone precipitation protocol encompasses significant differences over FASP II in terms of how proteins are processed. Unlike acetone, proteins should remain in solution throughout the FASP II proto- col, being concentrated as they are retained by the MWCO filter. Following acetone precipitation, our protocol relies on a combina- tion of urea as well as the digestion step itself to resolubilize and recover peptides. Considering the molecular weight distribution of identified peptides, a trend was noted wherein larger molecular weight peptides were observed following the acetone precipitation protocol (Fig. 7). Examining the number of missed cleavage sites for these peptides, the acetone protocol generates peptides with a higher degree of missed cleavages. From the list of uniquely identi- fied peptides, 38% of the peptides identified from acetone contained one or more missed cleavages. This doubled the number observed through FASP II (19.4%). An increase in the number of missed cleav- ages may also partially contribute to the larger number of peptide and protein identifications obtained through acetone precipitation.