Comparative Study of Workflows Optimized for In-gel, In-solution, and On-filter Proteolysis in the Analysis of Plasma Membrane Proteins

Abstract

Proteomic studies of plasma membrane proteins are challenged by the limited solubility of these proteins and the limited activity of proteolytic enzymes in solubilizing agents such as SDS. In this work, we have evaluated three bottom-up workflows to obtain tryptic peptides from plasma membrane proteins solubilized with 2% SDS. The workflows are: in-gel digestion, in-solution digestion, and on-filter digestion. The efficiencies of these strategies, optimized to employ different matrices for trypsin cleavage, were compared using a plasma membrane sample enriched from multiple myeloma cells using a nanoparticle pellicle. Based on the number of proteins identified, number of transmembrane proteins identified, hydrophobicity, and spectral count per protein, the workflow that uses in-gel digestion is the most advantageous approach for analysis of plasma membrane proteins.

Surfactant-Aided Precipitation/on-Pellet-Digestion (SOD) Procedure Provides Robust and Rapid Sample Preparation for Reproducible, Accurate and Sensitive LC/MS Quantification of Therapeutic Protein in Plasma and Tissues

“…FASP and in-gel-digestion resulted in much lower peptide recoveries than SOD or in-solution-digestion (p < 0.05); this problem is more pronounced when the level of the target mAb was low (e.g., 1 μg/mL, Figure 3A), which can be attributed to the peptide loss due to the absorption of peptides in the gel or filter. 17,40 In terms of the variation of the peptide recovery, the rank is in-geldigestion > FASP > SOD ≈ in-solution-digestion. The relatively large variations by in-gel-digestion and FASP likely root from the absorption of peptides on the gel/filters.…”

Enrichment of Plasma Membrane Proteins Using Nanoparticle Pellicles: Comparison between Silica and Higher Density Nanoparticles

“…We have undertaken to enrich the entire plasma membrane to analyze integral proteins and proteins from both the inner and outer surfaces, using pellicles formed with cationic nanoparticles. We are also optimizing a proteomic workflow to provide proteolysis of highly hydrophobic proteins (8). …”

Profiling of urinary proteins in Karan Fries cows reveals more than 1550 proteins

“…Due to absence of bovine urinary proteome database, results of this study were compared with the human urinary proteome prepared by AS: proteins precipitated by ammonium suphate precipitation; PS: proteins extracted by ProteoSpin column urine concentration kit and DF: diafiltered samples were directly used for proteome analysis without any precipitation. compiling three extensively studied databases representing a total of 3395 proteins [7,16,25]. Of these proteins, 315 proteins (Supplementary Table 5) identified in this study overlapped with human urinary protein database significantly expand the number of proteins (n = 1256) identified in bovine urine as shown in Fig.…”

“…In addition, the resolution of the extracted proteins was very poor. Specifically, it was difficult to re-solubilize the protein pellet obtained from acetone and methanol precipitation as reported earlier [25,26]. It has been reported that acetone precipitates some salts and other metabolic waste components along with proteins [26] resulting in poor quality 2DE.…”

“…Although a number of studies have been carried out on human urinary proteome [7,16,23,25], no comprehensive information is available on bovine urinary proteome to date. Thus, this study reports the first ever bovine urinary proteome in cow as well as a database of proteins.…”