There are some limitations to this protocol. The primary challenge is sensitivity of our mass spectrometer, which allowed us to sequence just three aptamers properly in this study. The instrumentation available to any lab will vary; we have used a high definition nanospray instrument, but other researchers may have access to more or less sensitive spectrometers. The size of the library is another consideration, since the number of variations increases exponentially with the number of variable positions and the possible variations at each site. This would similarly result in an exponential increase in materials needed for synthesis and time required to perform selection. Here, we have selected from 65,536 possible sequences, but results in our lab indicate that with our current capabilities, we could feasibly sort a library of up to 2 million variations (for example, ten positions with four possible variations would give 1,048,576 sequences). With more information about the target, or experimental structural data on the aptamer, it could be possible to more precisely choose which modifications to use, since they need not be restricted to U-I, U-Ph, and U-Vi. Another current limitation is the requirement for the target to be fluorescently labelled; this will make some targets more challenging than others, but the list of fluorophore-labelled proteins reported is growing steadily.