Amicon-adapted enhanced FASP: an in-solution digestion-based alternative sample preparation method to FASP

Abstract

Sample preparation is a crucial step for liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics. Sodium dodecyl sulfate (SDS) is a powerful denaturing detergent that allows for long-term preservation of protein integrity. However, as it inhibits trypsin and interferes with LC-MS/MS analyses, it must be removed from samples prior to these experiments. The Filter-Aided Sample Preparation (FASP) method is actually one of the preferred and simplest methods for such purpose. Nonetheless, there exist great disparities in the quality of outcomes when comparing FASP to other protocols depending on the authors, and recent reports have pointed to concerns regarding its depth of proteome coverage. To address these issues, we propose an Amicon-adapted in-solution-based enhanced FASP (eFASP) approach that relies on current best practices in comprehensive proteomics sample preparation. Human megakaryoblastic leukaemia cancer cells' protein extracts were treated in parallel with both Amicon-adapted eFASP and FASP, quantified for remaining SDS and then analyzed with a 1-hr gradient LC-MS/MS run. The Amicon-adapted eFASP utilizes a passivated low molecular weight cut-off Amicon filter, and incorporates a cleaning step with a high-content deoxycholate buffer and a 'one-step-two-enzymes' trypsin/Lys-C in-solution digestion. Amicon-adapted eFASP was found more reproducible and deepened proteome coverage, especially for membrane proteins. As compared to FASP, Amicon-adapted eFASP removed much of SDS from high-protein samples and reached a notable depth of proteome coverage with nearly 1,700 proteins identified in a 1 hr LC-MS/MS single-run analysis without prior fractionation. Amicon-adapted eFASP can therefore be regarded as a simple and reliable sample preparation approach for comprehensive proteomics.

Urinary Exosomal Cystatin C and Lipopolysaccharide Binding Protein as Biomarkers for Antibody−Mediated Rejection after Kidney Transplantation

“…Exosome fraction was lysed using lysis buffer (50 mM Tris-HCl (pH 8.5), 5% SDS) and quantified using BCA assay. A measure of 100 ug of exosome proteins was digested into peptides using the amicon-adapted enhanced FASP method [ 22 ], and salt was removed by the C18 desalting cartridge (Sep-Pak C18 1 cc, Waters, Milford, MA, USA). For details, refer to previously published papers [ 23 , 24 ].…”

Convergence of Plasma Metabolomics and Proteomics Analysis to Discover Signatures of High-Grade Serous Ovarian Cancer

“…At first, we depleted the high abundant plasma proteins by a Multiple Affinity Removal Column Human 14 (100 × 4.6 mm; MARS14, Agilent, CA, USA) column equipped in the HPLC systems. We digested the proteins to peptides by the amicon-adapted enhanced FASP method [95] and salts were removed sequentially by the C18 desalting cartridge (Sep-Pak C18 1 cc, Waters, USA). First, 40 µL of plasma was injected into the MARS14 depletion column in which the top 14 abundant proteins (albumin, IgA, IgG, IgM, a1-antitrypsin, a1-acid glycoprotein, apolipoprotein A1, apolipoprotein A2, complement C3, transferrin, a2-marcoglobulin, transthyretin, haptoglobin, and fibrinogen) were depleted.…”

Evaluation of Protein Purification Techniques and Effects of Storage Duration on LC-MS/MS Analysis of Archived FFPE Human CRC Tissues

“…Due to formalin-induced protein cross-linking, strong detergents such as sodium dodecyl sulfate (SDS) are required for total tissue solubilization and protein extraction from FFPE tissues [17][18][19]. However, SDS binds to amino acids and thereby changes the protein spatial conformational structure.…”

“…In addition, SDS alters the chromatographic separation of peptides and also interferes with electrospray ionization (ESI) mass spectrometry by dominating mass spectra and significantly suppressing analyte ion signals since it is readily ionizable and present in greater abundances than individual peptide ions. For these reasons, SDS must be completely depleted from a sample before enzymatic digestion and LC-ESI MS/ MS analysis [17][18][19][20]. However, SDS removal with minimal sample loss is a challenging task and several gel-free approaches have been proposed over the years.…”