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Proteomics – Proteomics and Systems Biology

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

by Nicole Unterlander & Alan A. Doucette

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.