Ionic liquid 1-butyl-3-methyl imidazolium tetrafluoroborate for shotgun membrane proteomics

Abstract

The solubility and digestion efficiency are two crucial factors that might affect the identification of integral membrane proteins (IMPs). In this work, 1% (v/v) ionic liquid (IL), 1-butyl-3-methyl imidazolium tetrafluoroborate (BMIM BF 4 ), added in NH 4 HCO 3 buffer (pH 8.3), was applied as a sample preparation buffer for IMPs analysis. Compared to the commonly used sodium dodecyl sulfate and methanol methods, the number of identified IMPs from rat brain by microcolumn reversed phase liquid chromatography (μRPLC)-electrospray ionization tandem mass spectrometry (ESI-MS/MS) was improved by over three times, which might be due to the fact that BMIM BF 4 offered high solubilizing ability for IMPs and good compatibility for tryptic digestion. Furthermore, compared to Rapigest and urea methods, with BMIM BF 4 method, the number of identified IMPs from rat brain could be improved 25% and 80%, respectively, which might be contributed to the good solubilizing ability and high thermal stability of such IL. With the sample treated by BMIM BF 4 method, by 2D-nanoSCX-RPLC-ESI-MS/MS, 1,450 non-redundant proteins and 7,978 unique peptides were identified from rat brain, and 418 proteins contained at least one predicted transmembrane domain, with false discovery rates of less than 1% for peptide identification, and at least two identified unique peptides per protein. All these results demonstrate that the BMIM BF 4 method is of high potential for the large-scale identification of IMPs.

An activity-maintaining sequential protein extraction method for bioactive assay and proteome analysis of velvet antlers

“…Finally, the residue was extracted with STD lysis buffer (4% SDS, 0.1 M Tris-HCl and 0.1 M DTT), to obtain Fraction D. Fraction A, B and C were filtrated by syringe filters and desalted using ultrafilters (10 kDa cut off) at 3000 Â g and 4 1C, then followed by lyophilization for further activity assay and proteomic analysis. The antler proteins obtained by three aqueous buffers were digested according to our recent work [16], and Fraction D was processed according to FASP method [17] for proteomic analysis. BCA Protein Assay was used to measure the protein concentrations of extracted fractions according to reference [18].…”

Prefractionation and separation by C8 stationary phase: Effective strategies for integral membrane proteins analysis

“…The solubilization and digestion of membrane proteins were performed according to our recent work [24] with slight modification. In brief, one aliquot of the membrane pellets (containing ∼100 g of proteins) was resuspended in 37.5 L of 20% (v/v) BMIM BF 4 in 100 mM NH 4 HCO 3 buffer (pH 8.3), and sonicated for 40 min, followed by heating at 90 • C for 20 min for denaturation.…”

“…However, the analysis of IMPs presents great challenges due to the hydrophobic nature [4][5][6][7]. Recently, much attention has been paid to the solubilization of IMPs, by using chaotropes [8][9][10], detergents [9][10][11][12][13][14][15][16][17], organic solvents [9,16,[18][19][20], organic acids [21][22][23] and ionic liquid [24]. Besides, the separation of hydrophobic peptides with high recovery also plays a crucial role in IMP identification.…”

“…In this work, firstly, ionic liquids (ILs) based protocol developed by our group [24] was used to solubilize the membrane protein pellet from rat hippocampus. Secondly, the prefractionation of membrane protein digests with a reverse phase trap column during desalting was performed to decrease the complexity of the sample.…”

Large‐scale N‐glycoproteome map of rat brain tissue: Simultaneous characterization of insoluble and soluble protein fractions

“…In our recent work, ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM BF 4 ), added in ammonium bicarbonate buffer, was exploited for protein solubilization. The high solubilizing ability and good compatibility for tryptic digestion rendered it in superiority for membrane proteome analysis [23]. However, its application in integral membrane glycoprotein study has not been performed.…”