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

Abstract

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.

Integrated platform with a combination of online digestion and 18O labeling for proteome quantification via an immobilized trypsin microreactor

“…As shown in Table 3, the results of the total sample preparation time, commonly quantified protein number, the average relative errors of quantified proteins, and the average RSD values between three technical replicates with the online platform were all much better than that of the offline method, suggesting improved sensitivity, quantitative accuracy, and reproducibility. The commonly quanti-fied protein number of the offline method is very low, but it is in the same magnitude compared with our previous result with a similar offline operation, 26 which might be caused by the sample loss and poor reproducibility of the multi-step offline operation. For 16 O and 18 O online labeled yeast sample at mixing ratios of 1 : 5 and 1 : 10, the measured ratios were 5.12 and 9.81, respectively, with average relative errors (RE) 27,28 of 9.9% and 11.8%, respectively (Fig.…”

“…Although 18 O labeling is more amenable for trace amounts of sample compared with other chemical labeling strategies, 12 it still needs too many artificial operations for digestion, labeling, and deactivating trypsin. The multi-step offline operations and unavoidable sample absorption on the tube can lead to sample loss, 26 making it still difficult for the identification of trace amounts of sample. In our strategy, all these offline operations can be integrated in the platform, making the platform more accessible for the quantitative analysis of trace amounts of sample.…”

Integrated proteomics sample pretreatment method based onSCX/SAX mixed packing

“…是在SISPROT技术基础上发展起来的一种方法; 该方 法将所有样品制备步骤整合到一个装有SAX离子交换 填料和C18膜的枪头中, 并在SAX填料上完成蛋白质 预富集、酶解和多肽分级操作 [10] . Zhao等人 [11]…”

Improving the Identification and Coverage of Plant Transmembrane Proteins in Medicago Using Bottom–Up Proteomics

“…A previous Medicago truncatula study utilized an 8 M urea-based approach to solubilize and identify a wide range of proteins from different tissues (Marx et al, 2016). Here, we established a non-detergent proteomic approach by using FA to substitute for detergents and 8 M urea (Zhao et al, 2013) for the solubilization of membrane samples of Medicago.…”