Improved disc SDS‐PAGE for extraction of low molecular weight proteins from serum

The low molecular weight proteins can provide a lot of valuable information of biomarkers. To study these proteins, the high abundance and high molecular weight proteins must be removed prior to analysis. In this work, a simple and inexpensive disc SDS‐PAGE to extract low molecular weight proteins from human serum and cutoff proteins larger than 30 kDa was developed. Some experimental conditions were examined. The experimental results obtained by plate SDS‐PAGE and MALDI‐TOF MS showed that the molecular weight of extracted proteins was about in the range from 0.3 to 28 kDa. Some experiments, including precipitation of proteins in organic solvents, SPE and cytochrome C test, were carried out and the experimental results demonstrated successful recovery of proteins/peptides with molecular weight from several hundreds of dalton to about 30 kDa. The experimental results obtained by plate SDS‐PAGE indicated the repeatability was satisfactory.     

Enrichment of low molecular weight serum proteins using acetonitrile precipitation for mass spectrometry based proteomic analysis

A rapid acetonitrile (ACN)-based extraction method has been developed that reproducibly depletes high abundance and high molecular weight proteins from serum prior to mass spectrometric analysis. A nanoflow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) multiple reaction monitoring (MRM) method for 57 high to medium abundance serum proteins was used to characterise the ACN-depleted fraction after tryptic digestion. Of the 57 targeted proteins 29 were detected and albumin, the most abundant protein in serum and plasma, was identified as the 20th most abundant protein in the extract. The combination of ACN depletion and one-dimensional nano-LC/MS/MS enabled the detection of the low abundance serum protein, insulin-like growth factor-I (IGF-I), which has a serum concentration in the region of 100 ng/mL. One-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the depleted serum showed no bands corresponding to proteins of molecular mass over 75 kDa after extraction, demonstrating the efficiency of the method for the depletion of high molecular weight proteins. Total protein analysis of the ACN extracts showed that approximately 99.6% of all protein is removed from the serum. The ACN-depletion strategy offers a viable alternative to the immunochemistry-based protein-depletion techniques commonly used for removing high abundance proteins from serum prior to MS-based proteomic analyses.     

Analysis of the human urine endogenous peptides by nanoparticle extraction and mass spectrometry identification

Peptides in urine are excreted by kidney from the blood and tissues, which are composed of a large amount of hormones, cytokines, regulatory factors and the metabolized fragments of proteins. The peptide distribution in urine will reflect the physiological and pathophysiological processes in body. In past, limited information was reported about the composition of the peptides in urine. One possible reason is that the peptides in urine are fairly low abundant and there are high concentrations of salts and organic metabolites in the urine. In this report, we extracted the peptides from human urine by highly ordered mesoporous silica particles with the pore size of 2 nm, which will exclude the high molecular weight proteins over 12 kDa. The extracted peptides were then separated into fractions according to their molecular weight by size exclusion chromatography. Each of the fractions was further analyzed by MALDI-TOF MS and μRPLC–MS/MS. Totally, 193 peptides were identified by two-dimensional SEC/μRPLC–MS/MS analysis. By analyzing the progenitor protein of the peptides; we found that two-thirds of the proteins differed from the reported urine proteome database, and the high abundant proteins in urine proteome were less detected in the urine peptidome. The developed extraction and separation methods were efficient for the profiling of the endogenous peptides in human urine. The peptidome in human urine was complementary to the human urinary proteome and may provide an emerging field for biomarker discovery.     

Proteomics and Detection of Uromodulin in First-time Renal Calculi Patients and Recurrent Renal Calculi Patients

Renal calculi disease or known as kidney stone disease is the most common urological disorder in both men and women, although it is more prevalent in men. The lifetime chance for an individual to develop renal calculi is ~10% whereas the risk of recurrence in a 10-year period is 74%. Therefore, a diagnostic tool for screening or detecting renal calculi is greatly needed. In this study, we analyze urinary protein profiles from patients with renal calculi for the first time (RC), healthy subjects (HS), and patients with recurrent renal calculi (RRC) to identify a biomarker for detecting the disease. Urinary proteins were isolated by salt precipitation and the proteins resolved by SDS-PAGE. Target proteins were analyzed with LC/MS/MS. Thirty-two proteins were identified from healthy subjects and patients. Uromodulin was the most abundant urinary protein in HS but was a very faint band if detected at all from those that formed renal calculi for the first time (p < 0.05). Yet the excreted levels of urinary uromodulin in RRC were similar to those of the HS suggesting that uromodulin is a reliable biomarker for only RC. In addition, a few immunoglobulins that were commonly found in the urine of both RC and RRC, which include Ig alpha heavy chain 1, Ig gamma-2 c region, Ig gamma-3 heavy chain disease protein, Ig heavy chain variable region, Ig heavy constant region gamma 4, and Ig heavy chain. Ig heavy chain Fab frag and antibody a5b7 chain B may serve as potential biomarkers for renal calculi disease.     

血清中低分子量蛋白质的提取和鉴定

采用改进的圆盘凝胶电泳提取人血清中低分子量蛋白质, 去除了血清中分子量大于3×104的蛋白质, 将提取的低分子量蛋白质热变性后直接在溶液中酶解成肽, 经液相色谱-质谱分析, 并进行Mascot数据库检索, 确认出人血清中97种蛋白质.     

Assessment of metals bound to marine plankton proteins and to dissolved proteins in seawater

Studies based on laser ablation–inductively coupled plasma-mass spectrometry (LA–ICP-MS) have been performed to assess metal bound to dissolved proteins and proteins from marine plankton after two-dimensional polyacrylamide gel electrophoresis (2D PAGE). Dissolved proteins were pre-concentrated from surface seawater (60 L) by tangential ultrafiltration with 10 kDa molecular weight cut-off (MWCO) membranes and further centrifugal ultrafiltration (10 kDa) before proteins isolation by methanol/chloroform/water precipitation. Proteins isolation from plankton was assessed after different trichloroacetic acid (TCA)/acetone and methanol washing stages, and further proteins extraction with a phenol solution. LA–ICP-MS analysis of the electrophoretic profiles obtained for dissolved proteins shows the presence of Cd, Cr, Cu, and Zn in five spots analyzed. These proteins exhibit quite similar molecular weights (within the 10–14 kDa range) and pIs (from 5.8 to 7.3). Cd, Cr, Cu, and Zn have also been found to be associated to proteins isolated from plankton samples. In this case, Cd has been found to be bound to proteins of quite different molecular weight (9, 13 and 22 kDa) and pIs (4.5, 5.2, 5.5, and 10). However, trace elements such as Cr, Cu and Zn appear to be mainly bound to plankton proteins of low molecular weight and variable pI.     

Investigation of methods suitable for the matrix-assisted laser desorption/ionization mass spectrometric analysis of proteins from ribonucleoprotein complexes

A variety of protein isolation and purification techniques for ribonucleoprotein (RNP) complexes were investigated for their compatibility with downstream analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Ribosomal proteins from Escherichia coli 70S ribosomes were obtained using methods such as phenol extraction and precipitation by organic solvents or acids. Under optimal conditions, more than 90% of the expected ribosomal proteins were detected in a single MALDI-MS experiment. The most effective approach combined ribosome denaturation by buffer exchange with acid precipitation of the ribosomal ribonucleic acids. An improved acid precipitation approach, involving the sequential additions of acetic and trifluoroacetic acid, yielded more complete protein coverage while minimizing loss of ion signal from lower molecular weight proteins. With phenol extraction, substantial gains in ion abundance of higher molecular weight proteins are noted, although some of the lower molecular weight proteins were not efficiently extracted. These results illustrate several effective approaches for protein isolation from protein complexes such as RNPs that are MALDI-MS compatible, and these approaches should extend the use of MALDI-MS for proteomics-based analyses of other protein-nucleic acid complexes.     

Isolation, primary structure characterization and identification of the glycosylation pattern of recombinant goldfish neurolin, a neuronal cell adhesion protein

Neurolin is a growth-associated cell surface glycoprotein from goldfish and zebra fish which has been shown to be involved in axonal path-finding in the goldfish retina and suggested to function as a receptor for axon guidance molecules. Being a member of the immunoglobulin superfamily of cell adhesion proteins, neurolin consists of five N-terminal extracellular immunoglobulin (Ig)-like domains, a transmembrane and a short cytoplasmatic domain. Repeated injections of polyclonal Fab fragments against neurolin and of monoclonal antibodies against either Ig domains cause path-finding errors and disturbance of axonal fasciculation. In order to obtain a complete structural characterization and a molecular basis for structure-function determination, recombinant neurolin with the complete extracellular part but lacking the transmembrane and cytoplasmatic domain was expressed in Chinese hamster ovary (CHO) cells (CHO-neurolin). The isolation of CHO-neurolin was carried out by Ni-affinity chromatography and subsequent high-performance liquid chromatography (HPLC). An exact molecular mass determination was obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and revealed 60.9 kDa, which suggested that approximately 10 kDa are due to glycosylation. The predicted molecular mass is 51.5 kDa, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) yielded an apparent molecular mass of 72 kDa. Gel shift assays using SDS-PAGE and Western blot analysis with anti-neurolin antibodies provided consistent molecular mass data. The complete primary structure and N-glycosylation patterns were identified using specific lectin assays, MALDI/MS peptide mapping analysis by proteolytic and in-gel digestion, electrospray ionization MS and MALDI/MS in combination with specific glycosidase degradation. HPLC isolation of glycosylated peptide fragments and MS after selective deglycosylation revealed heterogeneous glycosylations at all five N-glycosylation consensus sites. All attached N-glycans are of the complex type and show a mainly biantennary structure; they are fucosylated with alpha(2,3)-terminal neuraminic acid. These data serve as a first detailed model to characterize the molecular recognition structures exhibited by the extracellular domains.     

Unmasking low-abundance peptides from human blood plasma and serum samples by a simple and robust two-step precipitation/immunoaffinity enrichment method

Proteomic analysis of human plasma and serum for identifying and validating disease-specific marker proteins and peptides has one major drawback besides its unique advantage as a readily available sample source for diagnostic assays. This disadvantage is represented by the predominance of several high- and middle-abundant proteins, which clearly hamper identification and quantification approaches of potential and validated protein and peptide biomarkers, which are often of very low abundance. During the last decades, a significant number of depletion and enrichment techniques evolved to address these two issues. We present here a cost-effective and easy-to-use strategy for protein depletion comprising a thermal precipitation protocol followed by a two-step liquid/liquid precipitation as well as using an immunoaffinity chromatography method for the specific enrichment and isolation of the low-abundance polypeptide N-terminal pro-B-type natriuretic peptide and its precursor proBNP clinically used as biomarkers for the detection of severe human heart failure and related diseases. The applicability of this approach is shown by SDS -CGE, SDS-PAGE, electrochemiluminescence immunoassay and nano-LC ESI-MS/MS. Our thermal precipitation protocol followed by a two-step liquid/liquid precipitation could also serve as a potential depletion technique for the characterization of other low-abundance peptides and proteins.     

Characterization of sodium dodecylsulfate polyacrylamide gel electrophoresis-separated M. agalactiae membrane antigens by mass spectrometry

Mycoplasma membrane proteins are generally designated according to their apparent molecular weight measured by SDS-PAGE. Several results about mycoplasma membrane antigens are conflicting because some doubts are emerging about the accuracy of the method utilised to identify the antigens. Aim of this work, was to characterise proteins separated after sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)-mass spectrometry to allow an uncontroversial designation of the antigens. Fifteen proteins with molecular weights ranging from 15,000 to 80,000 Da had been excised from gel and their whole molecular weight and proteolytic pattern had been determined using MALDI-TOF. The peptide pattern obtained using trypsin digestion allowed us to identify LipA, P48, P59, P80 and P40. Some other proteins showed analogies to proteins of Mycoplasma genitalium or Mycoplasma pneumoniae the only Mycoplasmas completely sequenced. There wasn't a close correspondence between the SDS-PAGE apparent molecular weight (generally used to name the proteins), the gene derived calculated mass and the molecular weight of whole proteins measured by MALDI-TOF. Only micro sequence data obtained by MS/MS allowed us to identify LipC, described as one of the most important Mycoplasma agalactiae antigens. This protein was found in correspondence with the 50 kDa region, instead of the 25 kDa region, confirming a phenomenon that we previously described.     

Extensive fractionation and identification of proteins within nasal lavage fluids from allergic rhinitis and asthmatic chronic rhinosinusitis patients

Allergic rhinitis (AR), chronic rhinosinusitis (CRS), and asthma are prevalent airway diseases that can have a substantial impact on a patient's quality of life. MS analyses of biological fluids can effectively screen for proteins associated with disease processes, however, initial detection of diagnostic proteins is difficult due to protein complexity and dynamic range. To enhance the detection of lower abundance proteins, intact nasal lavage fluid (NLF) proteins from nonpolypoid AR and from asthmatic CRS patients were extensively fractionated prior to LC/MS/MS analysis. Pooled NLF samples were processed to remove low molecular weight molecules and high abundance plasma proteins. Anion exchange (AX) chromatography followed by RP‐LC further separated the remaining intact NLF proteins. The resulting fractions were digested with trypsin and the peptides analyzed by LC/MS/MS. Spectra were searched with MASCOT, SEQUEST, and X!Tandem to obtain peptide identifications and subsequently analyzed by Scaffold software to identify parent proteins with at least 99% confidence. The 197 identified proteins are compared to those previously cited in the literature and the workflow evaluated to determine the usefulness for the detection of lower abundance proteins. This is the first extensive list of NLF proteins generated from CRS patients with coexisting asthma.     

Nontargeted urine metabolomics analysis of the protective and therapeutic effects of Citri Reticulatae Chachiensis Pericarpium on high-fat feed-induced hyperlipidemia in rats

In this study, we focused on studying the changes in urine metabolites in hyperlipidemic rats using ultra-performance liquid chromatography coupled with quadrupole time-of-fight mass spectrometry (UPLC–Q-TOF/MS) and metabolomics, as well as the effect of Citri Reticulatae Chachiensis Pericarpium (CRCP) on hyperlipidemia. These urine samples were examined by UPLC–Q-TOF/MS to obtain MS data. The MS data were analyzed by principal component analysis and partial least squares-discriminant analysis to identify the differential metabolites. CRCP reduced the body weight and levels of triglycerides, total cholesterol and low-density lipoprotein cholesterol and abnormally decreased high-density lipoprotein cholesterol in hyperlipidemic rats, which were significantly raised by a high-fat diet. Twenty-seven potential biomarkers were identified within the complex sample matrix of urine. Fourteen biomarkers increased in the hyperlipidemia rats compared with normal rats. Meanwhile, 13 biomarkers decreased. CRCP reversed abnormal changes in biomarkers, including 5-l -glutamyl-taurine, 5-aminopentanoic acid, cis-4-octenedioic acid and 2-octenedioic acid. These biomarkers show that hyperlipidemia is related to the metabolic pathways of taurine and hypotaurine metabolism, fatty acid biosynthesis , and arginine and proline metabolism . CRCP mainly prevents hyperlipidemia by intervening in these metabolic pathways.     

Routine diagnosis with PhastSystem compared to conventional electrophoresis: automated sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining and western blotting of urinary proteins

The recent introduction of the PhastSystem, an automatic electrophoresis and staining system with precast gradient-gels, allows rapid and reproducible analysis of proteinuria in patients suffering from renal injury. A routine method for sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGE) and silver staining of unconcentrated urine specimens in the PhastSystem is described and compared to our conventional "macro"-method with self-cast SDS-polyacrylamide gradient gels. The method described for the PhastSystem using 0.3 microL sample volumes and an 8-25% polyacrylamide gradient gel leads to highly reproducible results within 1.5 h. Before electrophoresis urine specimens were neither concentrated nor dialyzed. Samples with a protein concentration exceeding 5 mg/mL had to be diluted 1:5 (v/v). Analysis and documentation of PhastGels appeared as easy as with our conventional SDS-PAGE. Protein bands could reliably be identified by Western blotting. Urine and serum proteins, separated in PhastGels, were electrophoretically transferred to nitrocellulose and detected with specific antibodies against human albumin, transferrin, alpha-1-antitrypsin and IgG. Comparison of several standard kits for molecular weight determination revealed considerable differences concerning the quality of protein separation patterns. Availability of precast gels and automatization of SDS-PAGE and staining allows easy standardization of urine SDS-PAGE among clinical routine laboratories.     

OFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins

In this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum-protein bonds has been developed. The method is based on the use of sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study of the reagents used for PAGE, for OFFGEL-IEF separation, and post-separation treatment of gels (such as enzymatic digestion and sample preparation for MS analysis) was tackled regarding their suitability for the identification of platinum binding proteins using standard proteins incubated with cisplatin. The distribution of platinum in high and low molecular weight fractions (separated by cut-off filters) was determined by ICP-MS, which allows evaluating platinum-protein bond stability under the conditions studied. SDS-PAGE in the absence of β-mercaptoethanol or dithiotreitol preserved the platinum-protein bonds. In addition, neither the influence of the electric field during the electrophoretic separation, nor the processes of fixing, staining and destaining of proteins in the gel did result in the loss of platinum from platinum binding proteins. SDS-PAGE under non-reducing conditions provides separation of platinum-binding proteins in very narrow bands with quantitative recoveries. Different amounts of platinum-bound proteins covering the range 0.3-2.0 μg were separated and mineralised for platinum determination, showing good platinum linearity. Limits of detection for a mixture of five standard proteins incubated with cisplatin were between the range of 2.4 and 13.9 pg of platinum, which were satisfactory for their application to biological samples. Regarding OFFGEL-IEF, a denaturing solution without thiourea and without dithiotreitol is recommended. The suitability of the OFFGEL-IEF for the separation of platinum binding proteins of a kidney cytosol was demonstrated.     

Identification of rat urinary glycoproteome captured by three lectins using gel and LC-based proteomics

Many different types of urine proteome studies have been done, but urine glycoprotein studies are insufficient. Therefore, we studied the glycoproteins from rat urine, which could be used to identify biomarkers in an animal model. First, urinary proteins were prepared by using the dialysis and lyophilizing methods from rat urine. Glycoproteins enriched with lectin affinity purification, concanavalin A, jacalin and wheat germ agglutinin from the urinary proteins were separated by means of reverse-phase fast protein LC (FPLC) or 1-D PAGE. Each FPLC fraction and 1-D PAGE gel band were trypsin-digested and analyzed by means of nanoLC-MS/MS. LC-MS/MS analyses were carried out by using linear ion trap MS. A total of 318 rat urinary glycoproteins were identified from the FPLC fractions and gel bands; approximately 90% of identified proteins were confirmed as glycoproteins in Swiss-Prot. Many glycoproteins, known as biomarkers, including C-reactive protein, uromodulin, amyloid beta A4 protein, alpha-1-inhibitor 3, vitamin D-binding protein, kallikrein 3 and fetuin-A were identified in this study. By studying urinary glycoproteins collected from rat, these results may help to assist in identifying urinary biomarkers regarding various types of disease models.     

Electrospray MS and MALDI imaging show that non‐specific lipid‐transfer proteins (LTPs) in tomato are present as several isoforms and are concentrated in seeds

Non‐specific lipid‐transfer proteins (nsLTPs) are major human allergens in many plant species, albeit their role in plant biochemistry is still undefined. They are found in many plant species, either as one or several isoforms according to the species, and usually they are found to concentrate in the outer part of the fruits. In this work, the characterization of tomato nsLTP isoforms was performed on the three main fractions of Piccadilly tomato fruit (peel, pulp and seeds) by using ultracentrifuge devices with molecular cut‐off able to retain proteins with molecular weight typical of plant LTPs. The isolated proteins were further analysed by LC‐MS, in order to investigate the occurrence and the localization of tomato LTP isoforms. The chromatographic retention times, the molecular masses, the presence of eight cysteine residues in their tertiary structures and the sequence information obtained by MS, although not complete yet, allowed us to identify four different LTP isoforms, not yet reported in the literature, which were found to be concentrated in the seed fractions. None of the molecular masses of these potential LTPs was already present in the UniProtKB/SwissProt database. MALDI imaging experiments confirmed their presence and main localization in seeds, although the actual data hinted at their presence around seeds, rather than exactly in them. These data hint to a complicated scenario concerning LTP proteins in tomato. Copyright © 2014 John Wiley & Sons, Ltd.     

An approach to remove alpha amylase for proteomic analysis of low abundance biomarkers in human saliva

Proteomic characterization of human whole saliva for the identification of disease-specific biomarkers is guaranteed to be an easy-to-use and powerful diagnostic tool for defining the onset, progression and prognosis of human systemic diseases and, in particular, oral diseases. The high abundance of proteins, mainly alpha amylase, hampers the detection of low abundant proteins appearing in the disease state and therefore should be removed. In the present study a 2-DE was used to analyze human whole saliva following the removal of alpha amylase by affinity adsorption to potato starch. After alpha amylase removal whole saliva was analyzed by SDS-PAGE showing at least sixfold removal efficiency and by an alpha amylase activity assay showing 97% reduced activity. MS identification of the captured alpha amylase after elution demonstrated specific removal; 2-DE analysis showed the selective removal of alpha amylase and consequently increased gel resolution. MS identification of protein spots in the 60 kDa area revealed 15 proteins, which were masked before alpha amylase removal. In conclusion, treatment of human whole saliva with an alpha amylase removal device increases gel resolution and enables a higher protein sample for analysis.     

Murine fecal proteomics: A model system for the detection of potential biomarkers for colorectal cancer

Tumor related products shed into the feces offer a potential source of biomarkers for the detection of colorectal cancer (CRC). Using SDS-PAGE followed by nanoflow reversed-phased LC–MS/MS to analyse fecal samples from Apc^Min/+ mice (that develop spontaneous multiple intestinal neoplasia with age) we have identified 336 proteins (115 proteins of murine origin, 201 from fecal bacteria, 18 associated with food intake and 2 of apparent parasitic origin). 75% of the murine proteins identified in this study are predicted to be extracellular or associated with the cell plasma membrane. Of these proteins, a number of the murine homologues of colorectal cancer associated proteins (CCAP) such as hemoglobin, haptoglobin, hemopexin, alpha-2-macroglobulin and cadherin-17 have been identified, demonstrating the potential of fecal proteomics for detecting potential biomarkers and paving the way for subsequent MS/MS based biomarker studies on similar human samples.     

非小细胞肺癌患者尿中Exosomes蛋白质组的差异表达分析

除血浆之外,尿液是另一种寻找潜在生物标志物的重要生物材料。本研究以200000×g超速离心法分离正常人和非小细胞肺癌(NSCLC)患者尿液中的Exosomes,运用1DSDS-PAGE对Exosomes蛋白质组进行分组,从电泳胶上切取正常组和疾病组的20～31kDa条带,胰蛋白酶酶解后,进行HPLC-CHIP-MS/MS分析,并通过UniProtKB/SWISS-PORT数据库搜索鉴定了24种蛋白质,其中在NSCLC患者尿液Exosomes蛋白质组中发现了8种差异表达蛋白,包括免疫球蛋白κ的3个片段、2种Ras相关蛋白、谷胱甘肽S转移酶A2、血清淀粉样P成分前体和磷脂酰乙醇胺结合蛋白1。     

Rapid, highly efficient extraction and purification of membrane proteins using a microfluidic continuous-flow based aqueous two-phase system

Membrane proteins play essential roles in regulating various fundamental cellular functions. To investigate membrane proteins, extraction and purification are usually prerequisite steps. Here, we demonstrated a microfluidic aqueous PEG/detergent two-phase system for the purification of membrane proteins from crude cell extract, which replaced the conventional discontinuous agitation method with continuous extraction in laminar flows, resulting in significantly increased extraction speed and efficiency. To evaluate this system, different separation and detection methods were used to identify the purified proteins, such as capillary electrophoresis, SDS-PAGE and nano-HPLC-MS/MS. Swiss-Prot database with Mascot search engine was used to search for membrane proteins from random selected bands of SDS-PAGE. Results indicated that efficient purification of membrane proteins can be achieved within 5-7s and approximately 90% of the purified proteins were membrane proteins (the highest extraction efficiency reported up to date), including membrane-associated proteins and integral membrane proteins with multiple transmembrane domains. Compared to conventional approaches, this new method had advantages of greater specific surface area, minimal emulsification, reduced sample consumption and analysis time. We expect the developed method to be potentially useful in membrane protein purifications, facilitating the investigation of membrane proteomics.