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

Zhao, Qun; Sun, Liangliang; Liang, Yu; Wu, Qi; Yuan, Huan; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

doi:10.1016/j.talanta.2011.11.035

Cited by 13 publications

(10 citation statements)

References 39 publications

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“…For IMP extraction, the procedure followed the previously described method with minor modifications. 31 The details of the extraction method are shown in the Supporting Information.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Biphasic Microreactor for Efficient Membrane Protein Pretreatment with a Combination of Formic Acid Assisted Solubilization, On-Column pH Adjustment, Reduction, Alkylation, and Tryptic Digestion

et al. 2013

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Combining good dissolving ability of formic acid (FA) for membrane proteins and excellent complementary retention behavior of proteins on strong cation exchange (SCX) and strong anion exchange (SAX) materials, a biphasic microreactor was established to pretreat membrane proteins at microgram and even nanogram levels. With membrane proteins solubilized by FA, all of the proteomics sample processing procedures, including protein preconcentration, pH adjustment, reduction, and alkylation, as well as tryptic digestion, were integrated into an "SCX-SAX" biphasic capillary column. To evaluate the performance of the developed microreactor, a mixture of bovine serum albumin, myoglobin, and cytochrome c was pretreated. Compared with the results obtained by the traditional in-solution process, the peptide recovery (93% vs 83%) and analysis throughput (3.5 vs 14 h) were obviously improved. The microreactor was further applied for the pretreatment of 14 μg of membrane proteins extracted from rat cerebellums, and 416 integral membrane proteins (IMPs) (43% of total protein groups) and 103 transmembrane peptides were identified by two-dimensional nanoliquid chromatography-electrospray ionization tandem mass spectrometry (2D nano-LC-ESI-MS/MS) in triplicate analysis. With the starting sample preparation amount decreased to as low as 50 ng, 64 IMPs and 17 transmembrane peptides were identified confidently, while those obtained by the traditional insolution method were 10 and 1, respectively. All these results demonstrated that such an "SCX-SAX" based biphasic microreactor could offer a promising tool for the pretreatment of trace membrane proteins with high efficiency and throughput.

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“…For IMP extraction, the procedure followed the previously described method with minor modifications. 31 The details of the extraction method are shown in the Supporting Information.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Biphasic Microreactor for Efficient Membrane Protein Pretreatment with a Combination of Formic Acid Assisted Solubilization, On-Column pH Adjustment, Reduction, Alkylation, and Tryptic Digestion

et al. 2013

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“…It should be pointed out that, the collected fractions of peptides from 8 M urea extraction and those from C12Im-Cl extraction were, respectively, separated on C18 and C8 capillary columns, since our previous study demonstrated that C8 column was beneficial to improve the separation efficiency of hydrophobic peptides. 34 By such a protocol, the totally required MS/MS running time for the single-run analysis was about 34 h, much faster than that in previous work (12 days). 23 In total, 202362 ± 685 peptidespectrum matches (PSMs) were obtained (FDR < 1%), and 70217 ± 37 peptides, corresponding to 8976 ± 33 proteins, representing 8313 ± 32 gene products, were identified.…”

Section: ■ Results and Discussionmentioning

confidence: 85%

In-Depth Proteome Coverage by Improving Efficiency for Membrane Proteome Analysis

et al. 2017

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Although great achievement has been made in the mapping of human proteome, the efficiency of sample preparation still needs to be improved, especially for membrane proteins. Herein, we presented a novel method to deepen proteome coverage by the sequential extraction of proteins using urea and 1-dodecyl-3- methylimidazolium chloride (C12Im-Cl). With such a strategy, the commonly lost hydrophobic proteins by 8 M urea extraction could be further recovered by C12Im-Cl, as well as the suppression effect of high abundance soluble proteins could be decreased. Followed by the in situ sample preparation and separation with different stationary phases, more than 9810 gene products could be identified, covering 8 orders of magnitude in abundance, which was, to the best of our knowledge, the largest data set of HeLa cell proteome. Compared with previous work, not only the number of proteins identified was obviously increased, but also the analysis time was shortened to a few days. Therefore, we expect that such a strategy has great potential applications to achieve unprecedented coverage for proteome analysis.

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“…The sample is passed through the SPE system and the hydrophobic analyte molecules are adsorbed on the sorbent by hydrophobic interaction. Silica‐based sorbents are generally modified with groups such as C8 and C18 and SPE systems employing these functionalities have been utilized for the desalting of abundant proteins , proteome of Xenopus laevis eggs and early stage embryos , human hepatoma cell line PLC/PRF/5 lysates , mouse adipose tissue , integral membrane proteins , and elucidation of unknown peaks found in chromatograms for dirithromycin analysis .…”

Section: Desaltingmentioning

confidence: 99%

Sample Clean‐up Strategies for ESI Mass Spectrometry Applications in Bottom‐up Proteomics: Trends from 2012 to 2016

2017

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Bottom-up proteomics is a mass spectrometric (MS)-based approach for the characterization of peptides obtained from in-solution protein digestion. MS is favored over other methods for peptide and protein analysis because of its better sensitivity and high throughput. Inorganic ions and surfactants present in the sample or produced during tryptic digestion are detrimental in MS analysis and affect the proteome data, thus sample preparation for removal of these unwanted components has become essential. Here, we review 48 research papers on strategies for removal of salts and surfactants (in particular, SDS) prior to ESI-MS analysis in bottom-up proteomics from 2012 to 2016. The strategies were mostly based on SPE and membrane-based filter-aided sample preparation for salt and SDS removal, respectively. Some known limitations of SPE and filter-aided sample preparation procedures are that they can be time consuming, laborious, and require the use of organic solvents before a concentrated extract suitable for analysis is obtained. The development of faster analytical methods by reducing the sample preparation time and thereby, increasing sample throughput, and in a solvent-less and membrane-less operation, is a significant contribution to proteome research.

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

Cited by 13 publications

References 39 publications

Biphasic Microreactor for Efficient Membrane Protein Pretreatment with a Combination of Formic Acid Assisted Solubilization, On-Column pH Adjustment, Reduction, Alkylation, and Tryptic Digestion

Biphasic Microreactor for Efficient Membrane Protein Pretreatment with a Combination of Formic Acid Assisted Solubilization, On-Column pH Adjustment, Reduction, Alkylation, and Tryptic Digestion

In-Depth Proteome Coverage by Improving Efficiency for Membrane Proteome Analysis

Sample Clean‐up Strategies for ESI Mass Spectrometry Applications in Bottom‐up Proteomics: Trends from 2012 to 2016

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