A two-stage spin cartridge for integrated protein precipitation, digestion and SDS removal in a comparative bottom-up proteomics workflow

2018

Electrophoresis

SDS plays a key role in proteomics workflows, including protein extraction, solubilization and mass-based separations (e.g. SDS-PAGE, GELFrEE). However, SDS interferes with mass spectrometry and so it must be removed prior to analysis. We recently introduced an electrophoretic platform, termed transmembrane electrophoresis (TME), enabling extensive depletion of SDS from proteins in solution with exceptional protein yields. However, our prior TME runs required 1 h to complete, being limited by Joule heating which causes protein aggregation at higher operating currents. Here, we demonstrate effective strategies to maintain lower TME sample temperatures, permitting accelerated SDS depletion. Among these strategies, the use of a magnetic stir bar to continuously agitate a model protein system (BSA) allows SDS to be depleted below 100 ppm (>98% removal) within 10 min of TME operations, while maintaining exceptional protein recovery (>95%). Moreover, these modifications allow TME to operate without any user intervention, improving throughput and robustness of the approach. Through fits of our time-course SDS depletion curves to an exponential model, we calculate SDS depletion half-lives as low as 1.2 min. This promising electrophoretic platform should provide proteomics researchers with an effective purification strategy to enable MS characterization of SDS-containing proteins.

Section: Introductionmentioning

confidence: 78%

Accelerated SDS depletion from proteins by transmembrane electrophoresis: Impacts of Joule heating

Unterlander

2018

Electrophoresis

“…A custom centrifugal filter device, previously described by our group was used to facilitate protein precipitation . The device houses a 0.2 μm Teflon membrane filter at the base of a 1 mL spin cartridge, which traps precipitated proteins.…”

Section: Methodsmentioning

confidence: 99%

“…Here, we expand the scope of TME for purification of simple and complex proteome mixtures, ahead of bottom‐up and top‐down MS analysis. To benchmark the performance of TME, protein recovery and residual SDS is compared to that of conventional FASP, as well as acetone precipitation, both of which are conducted using simple membrane‐based centrifugal filter devices. We further demonstrate a MWCO membrane filter to deplete SDS with recovery of intact proteins for subsequent LC/MS analysis.…”

Section: Introductionmentioning

confidence: 99%

Membrane‐Based SDS Depletion Ahead of Peptide and Protein Analysis by Mass Spectrometry

Unterlander

2018

Proteomics

Self Cite

SDS interferes with both bottom-up and top-down MS analysis, requiring removal prior to detection. Filter-aided sample preparation (FASP) is favored for bottom-up proteomics (BUP) while acetone precipitation is popular for top-down proteomics (TDP). We recently demonstrated acetone precipitation in a membrane filter cartridge. Alternatively, our automated electrophoretic device, termed transmembrane electrophoresis (TME), depletes SDS for both TDP and BUP studies. Here TME is compared to these two alternative methods of SDS depletion in both BUP and TDP workflows. To do so, a modified FASP method is described applicable to the SDS purification and recovery of intact proteins, suitable for LC/MS. All three methods reliably deplete >99.8% SDS. TME provide higher sample yields (average 90%) than FASP (55%) or acetone precipitation (57%), translating into higher total protein identifications (973 vs 877 FASP or 890 acetone) and higher spectral matches (2.5 times) per protein. In a top down workflow, each SDS-depletion method yields high-quality MS spectra for intact proteins. These results show each of these membrane-based strategies is capable of depleting SDS with high sample recovery and high spectra quality for both BUP and TDP studies.

“…Unfortunately, SDS brings challenges to the proteomics workflow (eg, SDS is incompatible with LC‐MS). Several simple devices have been established, including some of our own design, to address proteome processing including separation and SDS removal. The term “gadget” describes any small mechanical and/or electrical device designed to perform specific tasks.…”

Section: The Proteomics Facility: Instrumentationmentioning

confidence: 99%

“…Only then could the product be properly evaluated from a scientific standpoint. We eventually published on the capacity of our ProTrap XG cartridge to deplete SDS and recover proteins, although at this point we had been working on product development for well over 2 years …”

Section: … Vs Moldingmentioning

confidence: 99%

Developing front‐end devices for improved sample preparation in MS‐based proteome analysis

Nickerson

2020

J Mass Spectrom

Self Cite

Chemical analysis has long relied on instrumentation, from the simplest (eg, burets) to the more sophisticated (eg, mass spectrometers) to facilitate precision measurements. Regardless of their complexity, the development of a new instrumental device can be a valued approach to address problems in science. In this perspective, we outline the process of novel device design, from early phase conception to the manufacturing and testing of the tool or gadget. Focus is placed on the development of improved front‐end devices to facilitate protein sample manipulations ahead of mass spectrometry, which therefore augment the proteomics workflow. Highlighted are some of the many training secrets, choices, and challenges that are inherent to the often iterative process of device design. In hopes of inspiring others to pursue instrument design to address relevant research questions, we present a summary list of points to consider prior to innovating their own devices.