Abstract
We describe a 96-well plate compatible membrane-based proteomic sample processing method, which enables the complete processing of 96 samples (or multiples thereof) within a single workday. This method uses a large-pore hydrophobic PVDF membrane that efficiently adsorbs proteins, resulting in fast liquid transfer through the membrane and significantly reduced sample processing times. Low liquid transfer speeds have prevented the useful 96-well plate implementation of FASP as a widely used membrane-based proteomic sample processing method. We validated our approach on whole-cell lysate and urine and cerebrospinal fluid as clinically relevant body fluids. Without compromising peptide and protein identification, our method uses a vacuum manifold and circumvents the need for digest desalting, making our processing method compatible with standard liquid handling robots. In summary, our new method maintains the strengths of FASP and simultaneously overcomes one of the major limitations of FASP without compromising protein identification and quantification.
Highlights
From the ‡Proteomics Center and Department of Pathology and §Division of Emergency Medicine, Boston Children’s Hospital, Boston, Massachusetts, ¶Department of Pathology and F.M
filteraided sample processing method (FASP) versus MStern Blot—Filter-based sample processing in general and FASP in particular have established themselves as widely used proteomics sample processing/digestion methods due to their improved sensitivity and throughput when compared with gel-based digestion methods and due to their improved compatibility with salts, detergents, and other denaturants in comparison to conventional solution-based processing methods
Cellulose ultrafiltration membranes that are used for the FASP approach feature a pore size of 1–3 nm (10 to 30 kDa molecular weight cut offs (MWCO)), giving rise to this significant flow restriction
Summary
The fast liquid transfers through the PVDF membrane can be realized in practice and is the major source of time savings for the MStern blotting sample processing in comparison to FASP (Fig. 1B). Performance of MStern Blot—After establishing that using hydrophobic PVDF for membrane-based proteomic sample processing instead of hydrophilic-regenerated cellulose as in the case of FASP results in significant time-savings, we investigated the compatibility of adsorption of complex protein mixtures with tryptic digestion.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have