The application of a MiSeq sequencing platform, which is a bench-top high-throughput sequencer, allowed >1.3 Gb of high-quality sequencing data to be produced at a low cost in a short time.
More recently, bench-top high-throughput sequencing instruments such as the Roche/454 GS Junior (Roche, Basel, Switzerland), Illumina MiSeq and Ion Torrent PGM (Life Technologies) have been released, which are capable of sequencing several megabase to gigabase pairs in a single lane-sequencing run with enhanced speed and cost performance.
Turbo-coded bit-error-rate performance (top) and data throughput (bottom) as a function of MT azimuth direction inside a 120° sector.
Figure 12 Normalized throughput; top row: CCH (a) normalized throughput for L = 900 m (CCH), (b) normalized throughput for L = 500 m (CCH), (c) normalized throughput for L = 350 m (CCH); bottom row: SCH (d) normalized throughput for L = 900 m (SCH), (e) normalized throughput for L = 500 m (SCH), (f) normalized throughput for L = 350 m (SCH).
Now that available bioinformatic platforms can accommodate high throughput top down MS/MS analyses that result in a multitude of ion types, such as the diverse array of fragments that arise from UVPD, the potential for hybrid fragmentation of intact proteins is feasible even for complex mixtures.
We believe that this extra layer of complexity on top of high-throughput material experimentation is necessary to tackle the biological complexity and further advance the biomaterials field.
Thus far, high-throughput top down analysis of complex mixtures has rarely been reported, with a few exceptions.
Intact protein separation is a notoriously challenging part of high-throughput top down analysis, and is widely considered to be the bottleneck preventing top down proteomics from being more widely adopted.
While the three HCD settings tested by no means represent an exhaustive optimization procedure, they are an adequate parameter range for determining methods that will optimally dissociate the largest proportion of proteins encountered in a top down high-throughput analysis.
We applied this equation, along with several other energies (NCE of 20, 25, and 30), across the set of detected proteins from the PAO1 proteome during high-throughput Top Down proteome analysis.
In this case, it is the tradeoff between performance (e.g., the net throughput on top of the medium access control layer) and average power consumption that really matters.
