Evaluation of six sample preparation procedures for qualitative and quantitative proteomics analysis of milk fat globule membrane

Abstract

Proteomic analysis of membrane proteins is challenged by the proteins solubility and detergent incompatibility with MS analysis. No single perfect protocol can be used to comprehensively characterize the proteome of membrane fraction. Here, we used cow milk fat globule membrane (MFGM) proteome analysis to assess six sample preparation procedures including one in-gel and five in-solution digestion approaches prior to LC-MS/MS analysis. The largest number of MFGM proteins were identified by suspension trapping (S-Trap) and filter-aided sample preparation (FASP) methods, followed by acetone precipitation without clean-up of tryptic peptides method. Protein identifications with highest average coverage was achieved by Chloroform/MeOH, in-gel and S-Trap methods. Most distinct proteins were identified by FASP method, followed by S-Trap. Analyses by Venn diagram, principal-component analysis, hierarchical clustering and the abundance ranking of quantitative proteins highlight differences in the MFGM fraction by the all sample preparation procedures. These results reveal the biased proteins/peptides loss occurred in each protocol. In this study, we found several novel proteins that were not observed previously by in-depth proteomics characterization of MFGM fraction in milk. Thus, a combination of multiple procedures with orthologous properties of sample preparation was demonstrated to improve the protein sequence coverage and expression level accuracy of membrane samples.

Proteomic profiling of the thrombin-activated canine platelet secretome (CAPS)

“…Sample preparation of S-Trap micro spin column (ProtiFi, Huntington, NY, USA) was according to the vendor’s protocol and Zougman et al . [54, 55]. Four hundred microliter were taken from each sample.…”

“…Reconstitution volumes were varied (10–20 μL) for lyophilized tryptic peptides from gel segments based on their protein content. Peptides were analyzed by nano-LC-ESI MS/MS in single technical replicates [56] using an Orbitrap Fusion TM Tribrid TM (Thermo-Fisher Scientific, San Jose, CA) mass spectrometer equipped with a nanospray Flex Ion Source, and coupled with a Dionex UltiMate 3000 RSLCnano system (Thermo, Sunnyvale, CA) [54, 57]. Reconstituted peptides from individual gel segments and in-solution digests were injected (10–20 μL) onto a PepMap C-18 RP nano trapping column (5 μm, 100 μm i.d.…”

CDK2 kinase activity is a regulator of male germ cell fate

“…In-gel trypsin digestion of immunoprecipitated CDK2 protein was performed as described earlier (Yang et al 2007). The tryptic digests were subjected to nanoLC-ESI-MS/MS analysis on Orbitrap Fusion TM Tribrid TM (Thermo-Fisher Scientific, San Jose, CA) mass spectrometer equipped with a nanospray Flex Ion Source, and a Dionex UltiMate3000RSLCnano system (Thermo, Sunnyvale, CA) following a protocol described earlier (Yang et al 2018;Thomas et al 2017). The column was reequilibrated with 0.1% FA for 23 min prior to the next run.…”

Structure and chemistry of lysinoalanine crosslinking in the spirochaete flagella hook

“…The in-gel tryptic digests were reconstituted in 20 μL of 0.5% FA for nanoLC-ESI-MS/MS analysis, which was carried out using an Orbitrap FusionTM TribridTM (Thermo-Fisher Scientific, San Jose, CA) mass spectrometer equipped with a nanospray Flex Ion Source and coupled with a Dionex UltiMate3000RSLCnano system (Thermo, Sunnyvale, CA). 59,60 The gel extracted peptide samples (5-15 μL) were injected onto a PepMap C-18 RP nanotrapping column (5 μm, 100 μm i.d x 20 mm) at 20 μL/min flow rate for rapid sample loading and then separated on a PepMap C-18 RP nano column (2 μm, 75 μm x 25 cm) at 35 °C. The tryptic peptides were eluted in a 90 min gradient of 5% to 38% CAN in 0.1% formic acid at 300 nL/min, followed by a 7 min ramping to 90% ACN-0.1% FA and an 8 min hold at 90% ACN-0.1% FA.…”