MStern Blotting–High Throughput Polyvinylidene Fluoride (PVDF) Membrane-Based Proteomic Sample Preparation for 96-Well Plates

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. Mass spectrometry (MS)-based proteomics is moving increasingly into the translational and clinical research arena, where robust and efficient sample processing is of particular importance. The conventional sample processing methods in proteomics, namely SDS-PAGE, or in-solution-based sample processing, are slow and laborious and thus do not easily provide the reproducibility and throughput to meet current demands. A paradigm shift was the introduction of a filteraided sample processing method (FASP), which is initially described by Manza et al.(1) and then fully realized in practice by Wisniewski et al. (2). These filter-aided methods make use of ultrafiltration membranes with molecular weight cut offs (MWCO) in the 10 to 30 kDa range to efficiently remove small molecules and salts and to capture denatured proteins on a cellulose filter even if the molecular weight of the protein is much smaller than the nominal MWCO of the ultrafiltration membrane. Thus, the denaturation step is crucial to ensure that proteins much smaller than the nominal MWCO are efficiently retained by, e.g. a 10 kDa MWCO filter.In translational and clinical proteomics, which normally include large cohorts, the multititer-well plate is the preferred format for sample processing and storage. Although the application of FASP in the 96-well plate format has been described (3, 4), the major limitation of FASP in the 96-well plate is the much slower speed at which the 96-well plates have to be centrifuged: while a single ultrafiltration unit withstands up to 14,000 ϫ g, the 96-well plate format can only be centrifuged at g-forces of up to 2,200 ϫ g. This significantly lower g-force for 96-well plates results in a slow liquid transfer, which in turn considerably prolongs the required centrifugation times to hours instead of tens of minutes for the three to four necessary centrifugation steps (i) for the initial loading, reduction and alkylation, (ii) for the different washing steps, and (iii) for the elution (3).Independent of the format FASP is performed in, the conventional FASP also requires relative large volumes of high salt concentration for efficient elution of the tryptic peptid...

Filter-Based Protein Digestion (FPD): A Detergent-Free and Scaffold-Based Strategy for TMT Workflows

“…The cluster with the highest mean and median GRAVY score consisted of proteins that were relatively lower in abundance in the FPD (green), namely, hydrophobic proteins, for example membrane proteins. Such a trend has been reported previously in the comparison between FASP and MStern (5). We suggest the potential use of a mass spectrometry-compatible detergent, such as RapiGest or ProteoMax to enhance digestion, as commonly added in non-filter-based methods (20).…”

“…In addition, FASP has also been modified to accommodate multi-well formats. For example, MStern extended the sample preparation into a 96-well plate (5). Unlike previous methods, MStern used a PVDF membrane to decrease centrifugation time, therefore increasing throughput.…”

Quick 96FASP for high throughput quantitative proteome analysis

“…While system suitability assessment and experimental design need to be considered for a successful proteomic experiment [2-4], sample cleanup and protein digestion are two primary components of the analysis [1]. Traditional gel-based approaches to prepare samples are laborious and time-consuming [1, 5]. Gel-free approaches such as precipitation with organic solvents prior to in-solution digestion do not recover complete proteomes [6, 7].…”

“…A noted disadvantage of the reported 96FASP method is the lengthy filtration time [5, 27, 28], Each spin step in the protocol takes 45 to 90 min while single filter-based FASP can be completed in one fourth of the time. With over ten centrifugation steps in the entire workflow, experiments may not be completed in a one-workday time frame, thus reducing its broad appeal.…”

Reducing the cost of semi-automated in-gel tryptic digestion and GeLC sample preparation for high-throughput proteomics

“…Protocols have been proposed for automated digestion of samples on a filter [3], within gel pieces [4,5], in solution [6] and for sample cleanup [7]. These methods can improve sample reproducibility; however, this can come at an increased cost due to the need for specialized equipment, such as filtration plates [3,8,9], custom desalting plates [6], flowthrough reactors [4]or vacuum plates [5]. These increased costs may be a deterrent for the routine adoption of automation, or for large scale studies where they are most needed.…”