Efficient Human Plasma Proteome Profiling at up to 240 Samples Per Day

LC-MS-based proteomics stands as a vital tool for the analysis of intricate biological and clinical samples. Within the separation core, the LC column assumes a pivotal role by reducing sample complexity before its injection into the MS. The micro pillar array column (µPAC™) emerges as an innovative LC column featuring uniformly arranged pillars within a microfluidic channel, offering both highly efficient separation and minimal back pressure. In this study, we used a 5.5 cm long µPAC Neo High Throughput column that has channels filled with rectangular-shaped silicon pillars, coupled with HRAM mass spectrometry. Our goal was to expedite bottom-up proteomics profiling of neat human plasma samples while leveraging pillar arrays' benefits, such as high reproducibility and minimal sample carry-over.

Using a 3-minute gradient 240 SPD method, the amount of protein groups that could be identified from neat human plasma steadily increased according to the sample load, up to a load of 200 ng plasma on column, where a plateau of about 250 protein groups was achieved. Increasing beyond 200 ng didn't significantly improve protein identification, suggesting 200 ng is sufficient for favorable outcomes in this timeframe. This was also confirmed by checking the basepeak chromatograms and overlaying the different sample loads, where 200 ng was identified as the onset of overloading. Given the extremely high throughput of this method, fair quantitation could be achieved resulting in up to 205 protein groups quantified at CV below 20% for 500 ng of plasma analyzed in triplicate.

Similar observations were done across different SPD settings (170, 100), where increasing sample loading beyond 500 ng did not increase proteome depth. By extending the gradient length to respectively 5.5 and 11 min, increased depth and especially an increase in number of quantifyable proteins could be obtained. The µPAC Neo High Throughput column achieved a pragmatic compromise at 170 SPD, quantifiying 248 protein groups (below 20% CV). The impact of extending gradients beyond the 5.5 min used for the 170 SPD method appeared to be very limited, as barely any depth could be gained. Ongoing efforts are focusing on extended performance testing and comparative analysis of the susceptibility to sample related carry-over related to different LC and injection configurations.