An automated in‐gel digestion/iTRAQ‐labeling workflow for robust quantification of gel‐separated proteins

Simple protein separation by 1DE is a widely used method to reduce sample complexity and to prepare proteins for mass spectrometric identification via in‐gel digestion. While several automated solutions are available for in‐gel digestion particularly of small cylindric gel plugs derived from 2D gels, the processing of larger 1D gel‐derived gel bands with liquid handling work stations is less well established in the field. Here, we introduce a digestion device tailored to this purpose and validate its performance in comparison to manual in‐gel digestion. For relative quantification purposes, we extend the in‐gel digestion procedure by iTRAQ labeling of the tryptic peptides and show that automation of the entire workflow results in robust quantification of proteins from samples of different complexity and dynamic range. We conclude that automation improves accuracy and reproducibility of our iTRAQ workflow as it minimizes the variability in both, digestion and labeling efficiency, the two major causes of irreproducible results in chemical labeling approaches.     

Sensitivity of mass spectrometry analysis depends on the shape of the filtration unit used for filter aided sample preparation (FASP)

Efficient protein solubilization using detergents is required for in‐depth proteome analysis, but successful LC‐MS/MS analysis greatly depends on proper detergents removal. A commonly used sample processing method is the filter‐aided sample preparation (FASP), which allows protein digestion and detergent removal on the same filtration device. Many optimizations of the FASP protocol have been published, but there is no information on the influence of the filtration unit typology on the detergents removal. The aim of this study was to compare the performance of conic and flat bottom filtration units in terms of number of proteins identified by LC‐MS/MS. We have analyzed 1, 10 and 100 μg of total cell lysate prepared using lysis buffer with different SDS concentrations. We compared the FASP protocol using conic and flat bottom filtration units to ethanol precipitation method. Subsequently, we applied our most performant protocol to single murine pancreatic islet, and identified up to 2463 protein using FASP versus 1169 proteins using ethanol precipitation. We conclude that FASP performance depends strongly on the filter shape: flat bottom devices are better suited for low‐protein samples, as they allow better SDS removal leading to the identification of greater number of proteins.     

Labeling and Functionalizing Amphipols for Biological Applications

Amphipols (APols) are short amphipathic polymers developed as an alternative to detergents for handling membrane proteins (MPs) in aqueous solution. MPs are, as a rule, much more stable following trapping with APols than they are in detergent solutions. The best-characterized APol to date, called A8-35, is a mixture of short-chain sodium polyacrylates randomly derivatized with octylamine and isopropylamine. Its solution properties have been studied in detail, and it has been used extensively for biochemical and biophysical studies of MPs. One of the attractive characteristics of APols is that it is relatively easy to label them, isotopically or otherwise, without affecting their physical-chemical properties. Furthermore, several variously modified APols can be mixed, achieving multiple functionalization of MP/APol complexes in the easiest possible manner. Labeled or tagged APols are being used to study the solution properties of APols, their miscibility, their biodistribution upon injection into living organisms, their association with MPs and the composition, structure and dynamics of MP/APol complexes, examining the exchange of surfactants at the surface of MPs, labeling MPs to follow their distribution in fractionation experiments or to immobilize them, increasing the contrast between APols and solvent or MPs in biophysical experiments, improving NMR spectra, etc. Labeling or functionalization of APols can take various courses, each of which has its specific constraints and advantages regarding both synthesis and purification. The present review offers an overview of the various derivatives of A8-35 and its congeners that have been developed in our laboratory and discusses the pros and cons of various synthetic routes.     

Long-Term Stability of a Vaccine Formulated with the Amphipol-Trapped Major Outer Membrane Protein from Chlamydia trachomatis

Chlamydia trachomatis is a major bacterial pathogen throughout the world. Although antibiotic therapy can be implemented in the case of early detection, a majority of the infections are asymptomatic, requiring the development of preventive measures. Efforts have focused on the production of a vaccine using the C. trachomatis major outer membrane protein (MOMP). MOMP is purified in its native (n) trimeric form using the zwitterionic detergent Z3–14, but its stability in detergent solutions is limited. Amphipols (APols) are synthetic polymers that can stabilize membrane proteins (MPs) in detergent-free aqueous solutions. Preservation of protein structure and optimization of exposure of the most effective antigenic regions can avoid vaccination with misfolded, poorly protective protein. Previously, we showed that APols maintain nMOMP secondary structure and that nMOMP/APol vaccine formulations elicit better protection than formulations using either recombinant or nMOMP solubilized in Z3–14. To achieve a greater understanding of the structural behavior and stability of nMOMP in APols, we have used several spectroscopic techniques to characterize its secondary structure (circular dichroism), tertiary and quaternary structures (immunochemistry and gel electrophoresis) and aggregation state (light scattering) as a function of temperature and time. We have also recorded NMR spectra of ¹⁵N-labeled nMOMP and find that the exposed loops are detectable in APols but not in detergent. Our analyses show that APols protect nMOMP much better than Z3–14 against denaturation due to continuous heating, repeated freeze/thaw cycles, or extended storage at room temperature. These results indicate that APols can help improve MP-based vaccine formulations.     

Comparison of detergent‐based sample preparation workflows for LTQ‐Orbitrap analysis of the Escherichia coli proteome

This work presents a comparative evaluation of several detergent‐based sample preparation workflows for the MS‐based analysis of bacterial proteomes, performed using the model organism Escherichia coli. Initially, RapiGest‐ and SDS‐based buffers were compared for their protein extraction efficiency and quality of the MS data generated. As a result, SDS performed best in terms of total protein yields and overall number of MS identifications, mainly due to a higher efficiency in extracting high molecular weight (MW) and membrane proteins, while RapiGest led to an enrichment in periplasmic and fimbrial proteins. Then, SDS extracts underwent five different MS sample preparation workflows, including: detergent removal by spin columns followed by in‐solution digestion (SC), protein precipitation followed by in‐solution digestion in ammonium bicarbonate or urea buffer, filter‐aided sample preparation (FASP), and 1DE separation followed by in‐gel digestion. On the whole, about 1000 proteins were identified upon LC‐MS/MS analysis of all preparations (>1100 with the SC workflow), with FASP producing more identified peptides and a higher mean sequence coverage. Each protocol exhibited specific behaviors in terms of MW, hydrophobicity, and subcellular localization distribution of the identified proteins; a comparative assessment of the different outputs is presented.     

Quantitative Assessment of In-solution Digestion Efficiency Identifies Optimal Protocols for Unbiased Protein Analysis

The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative liquid chromatography-tandem MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative liquid chromatography-tandem MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows for efficient, unbiased generation and recovery of peptides from all protein classes, including membrane proteins. This deoxycholate-assisted protocol was also optimal for spin filter-aided digestions as compared with existing methods.MS-based proteomics is an indispensable technology for the characterization of complex biological systems, including relative or absolute protein expression levels and protein post-translational modifications. The most popular method for analyzing medium to high complexity protein samples in large-scale proteomics relies on protein digestion by using the endoprotease trypsin. Analysis and sequencing of tryptic peptides by liquid chromatography-tandem MS (LC-MS/MS)¹ then enables identification and determination of protein expression levels based on the peptide ion abundance level or the (fragment) ion intensities of identified peptides. This peptide-centric approach thus strongly relies on efficient, unbiased and reproducible protein digestion protocols. Efficiency is required to maximize the number of detectable peptides per protein (coverage) to distinguish unique proteins within protein families with similar sequences and/or sequence variants, and to detect post-translational modifications. Unbiased generation of peptides is required for the resulting data set to most accurately reflect the relative (stoichiometry) and absolute protein abundance in a sample. A particular protocol should be unbiased with respect to abundance, molecular weight, hydrophobicity and protein class. Membrane proteins for example are often suspected to be underrepresented. For MS-based proteomics approaches several critical steps can be distinguished: (a) disruption and solubilization of cells and protein complexes, (b) protein denaturation and enzymatic proteolysis, (c) MS-compatible peptide recovery, which normally entails removal of reagent leftovers and desalting before MS analysis, (d) adequate peptide separation (achieved by liquid chromatography), and (e) MS peptide analysis and sequencing (MS/MS), including the chosen data acquisition strategy.Comparative evaluations of digestion protocols generally consist of qualitative studies using standard tandem mass spectrometry. These approaches may reveal efficiency (i.e. more identifications), but are unable to reveal digestion protocol induced bias with respect to peptide and protein abundance, including membrane proteins. In addition, most data-dependent acquisition workflows are intrinsically biased, which is detrimental for making comparisons. The aim of the present study was to systematically assess efficiency and bias of trypsin-based protocols applying both standard qualitative and label-free quantitative MS approaches.The in-gel digestion protocol for proteomics, established over 15 years ago (1), has been the cornerstone method affording robust protein identifications from many sample types. Although sodium dodecyl sulfate (SDS) interferes with trypsin digestion and hampers LC-MS analysis, this powerful detergent can still be used to achieve complete protein solubilization as gel-separation is an effective way to remove interfering substances. Gel-based approaches are however not optimal for protein samples of increasing complexity and dynamic range (2). Inherent and practical limitations include, for example, concentration-dependent, incomplete peptide recovery and error-prone handling procedures (3–6). This hampers throughput, reproducibility and unbiased protein analysis, which in recent years has prompted a shift toward the application and optimization of in-solution digestion procedures.Previous comparative studies revealed that for in-solution digestions, the acid labile and MS-compatible detergent RapiGest performed most favorably compared with buffer only, urea, other detergents and organic solvents (7–9). Sodium deoxycholate (SDC), naturally found in mammalian bile (10), has emerged as a cheaper MS-compatible detergent for in-solution digestion (11). Unlike other detergents, SDC was found to enhance trypsin activity almost fivefold at a concentration of 1% (12). Like RapiGest, SDC can also be removed by acidification, but potentially without detrimental peptide loss if a phase separation protocol involving organic solvent is applied (12).An alternative strategy is to perform protein digestion on spin filter devices, introduced a few years ago by Manza and co-workers (13), and further developed by Wisniewski et al. (14). This approach allows the use of SDS to first achieve complete protein solubilization followed by removal of the detergent through repeated washes with urea (14). This is an effective way to remove interfering chemicals and small molecules after protein solubilization, and before digestion, without substantial sample loss. Although this protocol is touted to be a highly effective and universal method for any type of sample, digestion is performed using urea or buffer only and has so far not been evaluated in combination with detergents such as SDC.For our comparative study we selected protocols and methods based on spin filter-aided and standard in-solution digestion that were previously reported optimal and we also report novel optimized protocols. We investigated several experimental parameters including reagents for protein solubilization and denaturation (SDS, SDC, urea), spin filter aided removal of SDS before digestion (urea, SDC, buffer), trypsin digestion conditions (buffer, RapiGest, SDC, urea), and methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate).Mitochondria are organelles carrying out key metabolic processes fundamental for cellular function (15). The mitochondrial proteome is predicted to contain up to a thousand proteins (16) and is very heterogeneous with a wide range of protein pI, molecular weight and hydrophobicity values (17). We selected mitochondrial preparations to serve as model sample of medium complexity, containing a favorable combination of peptide and protein classes, including soluble and insoluble membrane-anchored or integral proteins.Using standard qualitative as well as data-independent quantitative LC-MS/MS workflows we demonstrate that SDC-based protocols combined with phase separation are the most optimal for both in-solution and filter-aided tryptic digestion, yielding the highest efficiency and lowest bias. This workflow enabled quantitative and objective assessment of various protein digestion conditions, identifying optimal protocols for efficient and unbiased protein analysis.     

Comparative study of workflows optimized for in-gel, in-solution, and on-filter proteolysis in the analysis of plasma membrane proteins

Proteomic studies of plasma membrane proteins are challenged by the limited solubility of these proteins and the limited activity of proteolytic enzymes in solubilizing agents such as SDS. In this work, we have evaluated three bottom-up workflows to obtain tryptic peptides from plasma membrane proteins solubilized with 2% SDS. The workflows are in-gel digestion, in-solution digestion, and on-filter digestion. The efficiencies of these strategies, optimized to employ different matrices for trypsin cleavage, were compared using a plasma membrane sample enriched from multiple myeloma cells using a nanoparticle pellicle. On the basis of the number of proteins identified, number of transmembrane proteins identified, hydrophobicity, and spectral count per protein, the workflow that uses in-gel digestion is the most advantageous approach for analysis of plasma membrane proteins.     

Amphipols for Each Season

Amphipols (APols) are short amphipathic polymers that can substitute for detergents at the transmembrane surface of membrane proteins (MPs) and, thereby, keep them soluble in detergent free aqueous solutions. APol-trapped MPs are, as a rule, more stable biochemically than their detergent-solubilized counterparts. APols have proven useful to produce MPs, most noticeably by assisting their folding from the denatured state obtained after solubilizing MP inclusion bodies in either SDS or urea. They facilitate the handling in aqueous solution of fragile MPs for the purpose of proteomics, structural and functional studies, and therapeutics. Because APols can be chemically labeled or functionalized, and they form very stable complexes with MPs, they can also be used to functionalize those indirectly, which opens onto many novel applications. Following a brief recall of the properties of APols and MP/APol complexes, an update is provided of recent progress in these various fields.     

A bioinformatics workflow for variant peptide detection in shotgun proteomics

Shotgun proteomics data analysis usually relies on database search. However, commonly used protein sequence databases do not contain information on protein variants and thus prevent variant peptides and proteins from been identified. Including known coding variations into protein sequence databases could help alleviate this problem. Based on our recently published human Cancer Proteome Variation Database, we have created a protein sequence database that comprehensively annotates thousands of cancer-related coding variants collected in the Cancer Proteome Variation Database as well as noncancer-specific ones from the Single Nucleotide Polymorphism Database (dbSNP). Using this database, we then developed a data analysis workflow for variant peptide identification in shotgun proteomics. The high risk of false positive variant identifications was addressed by a modified false discovery rate estimation method. Analysis of colorectal cancer cell lines SW480, RKO, and HCT-116 revealed a total of 81 peptides that contain either noncancer-specific or cancer-related variations. Twenty-three out of 26 variants randomly selected from the 81 were confirmed by genomic sequencing. We further applied the workflow on data sets from three individual colorectal tumor specimens. A total of 204 distinct variant peptides were detected, and five carried known cancer-related mutations. Each individual showed a specific pattern of cancer-related mutations, suggesting potential use of this type of information for personalized medicine. Compatibility of the workflow has been tested with four popular database search engines including Sequest, Mascot, X!Tandem, and MyriMatch. In summary, we have developed a workflow that effectively uses existing genomic data to enable variant peptide detection in proteomics.     

Dye-promoted precipitation of serum proteins. Mechanism and application.

Immobilized dyes have been used primarily for purification of nucleotide dependent enzymes and proteins from plasma and other sources. Due to their low costs, high protein binding capacity and resistance to degradation dyes bear the potential as ligand for affinity separation of proteins on a large scale. In this paper dyes have been used for precipitation of proteins. Using albumin, prealbumin, alpha 1-acid glycoprotein and immunoglobulin G as model proteins we could demonstrate that dye-promoted precipitation depends on several factors which include the structure of the dye, the pH of the solution, the dye/protein molar ratio and the intrinsic properties of the proteins. It revealed that most of the dyes tested were endowed with the precipitating potential. The efficacy of precipitation was found to increase with the complexity of the dye structure. However, the amount of a dye required for total precipitation was found to be different for a given protein. Electrostatic as well as hydrophobic forces are involved in the mechanism of precipitation. It was demonstrated that by optimizing the conditions, mixtures of proteins can be resolved by dye-promoted precipitation. The high sensitivity of the reaction offers the possibility of using this method for rapid concentration of very diluted protein solutions.     

Extraction method for analysis of detergent-solubilized bacteriorhodopsin and hydrophobic peptides by electrospray ionization mass spectrometry

The analysis of integral membrane proteins or transmembrane peptides by electrospray ionization mass spectrometry (ESI-MS) is difficult since detergents, used to solubilize these hydrophobic proteins and peptides, severely suppress analyte ion formation. This problem has been addressed previously by precipitating the protein, removing the detergent, and resolubilizing the protein in a nonpolar solvent. Here, we demonstrate a method that avoids protein precipitation and resolubilization. Detergent-solubilized bacteriorhodopsin is extracted into a nonpolar solvent phase by adding a chloroform/methanol/water solvent mixture to the aqueous detergent solution. ESI mass spectra of the nonpolar, chloroform-rich phase were dominated by peaks due to bacterioopsin. Bacterioopsin precursors with partially cleaved leader sequences were seen in all mass spectra. Additional peaks were likely due to intact bacteriorhodopsin, i.e., bacterioopsin with the retinal prosthetic group attached, and to bacterioopsin associated with lipid molecules. A separation process that occurred in the fused-silica capillary leading to the electrospray tip was essential for obtaining ESI mass spectra of bacterioopsin. The extraction-into-chloroform procedure also worked well with hydrophobic, transmembrane-type peptides that were insoluble in other electrospray solvents, including 100% formic acid, and the method has application to transmembrane peptides formed from digests of integral membrane proteins.     

Gel absorption-based sample preparation for the analysis of membrane proteome by mass spectrometry

A gel absorption-based sample preparation method for shotgun analysis of membrane proteome has been developed. In this new method, membrane proteins solubilized in a starting buffer containing a high concentration of sodium dodecyl sulfate (SDS) were directly entrapped and immobilized into gel matrix when the membrane protein solution was absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts were removed by washing, the proteins were subjected to in-gel digestion and the tryptic peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). The results showed that the newly developed method not only avoided the protein loss and the adverse protein modifications during gel embedment but also improved the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides, thereby facilitating the identification of membrane proteins, especially the integral membrane proteins. Compared with the conventional tube-gel digestion method, the newly developed method increased the numbers of identified membrane proteins and integral membrane proteins by 25.0% and 30.2%, respectively, demonstrating that the method is of broad practicability in gel-based shotgun analysis of membrane proteome.     

Evaluation of the application of sodium deoxycholate to proteomic analysis of rat hippocampal plasma membrane

Detergents have been widely used for the solubilization of membrane proteins and the improvement of their digestion. In this paper, we have evaluated the application of sodium deoxycholate (SDC) to the solubilization and digestion of rat hippocampal plasma membrane (PM) proteins. For in-solution digestion, rat hippocampal PM fraction from sucrose-density gradient centrifugation was solubilized by boiling in 1.0% SDC, and directly digested without dilution. During the in-gel digestion of the hippocampal PM proteins separated by SDS-PAGE, 0.1% SDC was added. Before analysis of peptide mixture by liquid chromatography and electrospray mass spectrometry, SDC in the tryptic digests was removed by centrifugation following acidification. Use of 1.0% SDC in solubilization and in-solution digestion of rat PM proteins had led to 77 PM or membrane-associated proteins identified, a more than 2-fold increase over that by use of SDS. The addition of 0.1% SDC to the in-gel digestion of SDS-PAGE-resolved membrane proteins remarkably enhanced the coverage of tryptic peptides and the number of hydrophobic membrane proteins identified. Being a cheaper and more tractable acid-insoluble detergent, SDC could be used at higher concentration in the solubilization and tryptic digestion of proteins including PM proteins with the purpose of enhancing the protein solubility and at the same time making no interference with trypsin activity and subsequent analyses.     

Label-free mass spectrometry-based relative quantification of proteins separated by one-dimensional gel electrophoresis

Here we present a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI–TOF/TOF)-based label-free relative protein quantification strategy that involves sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) separation of proteins followed by in-gel trypsin digestion. The main problem encountered in gel-based protein quantification is the difficulty in achieving complete and consistent proteolytic digestion. To solve this problem, we developed a high-pressure-assisted in-gel trypsin digestion method that is based on pressure cycling technology (PCT). The PCT approach performed at least as well as the conventional overnight in-gel trypsin digestion approach in parameters such as number of peaks detected, number of peptides identified, and sequence coverage, and the digestion time was reduced to 45 min. The gel/mass spectrometry (MS)-based label-free protein quantification method presented in this work proved the applicability of the signal response factor concept for relative protein quantification previously demonstrated by other groups using the liquid chromatography (LC)/MS platform. By normalizing the average signal intensities of the three most intense peptides of each protein with the average intensities of spiked synthetic catalase tryptic peptides, which we used as an internal standard, we observed spot-to-spot and lane-to-lane coefficients of variation of less than 10 and 20%, respectively. We also demonstrated that the method can be used for determining the relative quantities of proteins comigrating during electrophoretic separation.     

Protein precipitation of diluted samples in SDS‐containing buffer with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach

Proteomic approaches are extremely valuable in many fields of research, where mass spectrometry methods have gained an increasing interest, especially because of the ability to perform quantitative analysis. Nonetheless, sample preparation prior to mass spectrometry analysis is of the utmost importance. In this work, two protein precipitation approaches, widely used for cleaning and concentrating protein samples, were tested and compared in very diluted samples solubilized in a strong buffer (containing SDS). The amount of protein recovered after acetone and TCA/acetone precipitation was assessed, as well as the protein identification and relative quantification by SWATH‐MS yields were compared with the results from the same sample without precipitation. From this study, it was possible to conclude that in the case of diluted samples in denaturing buffers, the use of cold acetone as precipitation protocol is more favourable than the use of TCA/acetone in terms of reproducibility in protein recovery and number of identified and quantified proteins. Furthermore, the reproducibility in relative quantification of the proteins is even higher in samples precipitated with acetone compared with the original sample.     

基于超滤辅助样品制备方法的优化简

目的：基于超滤辅助样品制备（FASP）方法的出现使得使用去污剂（如SDS）的蛋白质提取方法与溶液内酶切方法得以兼容,因此提高了难溶性蛋白的鉴定数量。然而,超滤膜的非特异性吸附作用依然会造成蛋白的损失。我们拟针对该方法存在的问题对其进行改进。方法：对FASP方法进行了蛋白酶切条件、洗脱液选择、洗脱次数的改进;为测试优化方法的有效性和适用范围,选择标准蛋白BSA、鼠肝和鼠脑等3种样品进行考察。结果：相比报道的FASP方法,采用改进后的FASP方法使BSA的回收率提高了20％;经高精度质谱检测,对鼠肝、鼠脑的蛋白质鉴定结果分别比采用未优化的FASP多鉴定到2086和3592条特异肽段。结论：通过对FASP方法的优化,蛋白鉴定数量得到较大提高,该方法为蛋白质组深度覆盖研究提供了可靠的技术手段。     

Efficient removal of detergents from proteins and peptides in a spin column format

Detergents are commonly used in protein–chemistry protocols and may be necessary for protein extraction, solubilization, and denaturation; however, their presence interferes with many downstream analysis techniques, including mass spectrometry (MS). To enable downstream analysis, it is critical to remove unbound detergents from protein and peptide samples. In this study, we describe a high-performance resin that offers exceptional detergent removal for proteins and peptides. When used in a spin column format, this resin dramatically improves protein and peptide MS results by more than 95% removal of 1–5% detergents, including sodium dodecyl sulfate (SDS), sodium deoxycholate, Chaps, Triton X-100, Triton X-114, NP-40, Brij-35, octyl glucoside, octyl thioglucoside, and lauryl maltoside, with high recovery of proteins and peptides. Postcolumn liquid chromatography–tandem MS (LC–MS/MS) analysis of trypsin digests of bovine serum albumin (BSA) and HeLa cell lysate revealed excellent sequence coverage, indicating successful removal of detergent from the peptides. Matrix-assisted laser desorption/ionization (MALDI)–MS analysis of unprocessed and processed samples further confirmed efficient removal of detergents. The advantages of this method include speed (<15 min), efficient detergent removal, and high recovery of proteins and peptides.     

Solution Behavior and Crystallization of Cytochrome bc 1 in the Presence of Amphipols

Detergents classically are used to keep membrane proteins soluble in aqueous solutions, but they tend to destabilize them. This problem can be largely alleviated thanks to the use of amphipols (APols), small amphipathic polymers designed to substitute for detergents. APols adsorb at the surface of the transmembrane region of membrane proteins, keeping them water-soluble while stabilizing them bio-chemically. Membrane protein/APol complexes have proven, however, difficult to crystallize. In this study, the composition and solution properties of complexes formed between mitochondrial cytochrome bc ₁ and A8-35, the most extensively used APol to date, have been studied by means of size exclusion chromatography, sucrose gradient sedimentation, and small-angle neutron scattering. Stable, monodisperse preparations of bc ₁/A8-35 complexes can be obtained, which, depending on the medium, undergo either repulsive or attractive interactions. Under crystallization conditions, diffracting three-dimensional crystals of A8-35-stabilized cytochrome bc ₁ formed, but only in the concomitant presence of APol and detergent.     

Efficient digestion and mass spectral analysis of vesicular glutamate transporter 1: a recombinant membrane protein expressed in yeast

Attempts to characterize recombinant integral membrane proteins (IMPs) by mass spectrometry are frequently hindered by several factors including the detergents required for extraction and purification that interferes with analysis, poor solubility, incomplete digestion, and limited identification of the transmembrane domain-spanning peptides. The goal of this study was to examine and develop methods for purification of an IMP that are amenable to downstream digestion of the protein and peptide analysis by mass spectrometry. In this study, we have overexpressed a candidate IMP, the vesicular glutamate transporter 1 (VGLUT1) in Pichia pastoris and examined conditions for the efficient affinity purification, in-solution digestion, and analysis of the protein. Analysis of the intact purified protein without detergent was performed by MALDI-TOF mass spectrometry. The purified IMP was digested with trypsin, and the resulting peptides were identified. A method that utilizes differential solubility and ionization properties of hydrophobic and hydrophilic peptides was developed. Large hydrophobic peptides were only detected in solutions containing 50% formic acid. Ionization of hydrophilic peptides was suppressed in formic acid, but they produced a strong signal in 50% acetonitrile. Eighty-seven percent sequence coverage of the protein was obtained with only one large hydrophobic peptide that remained unidentified. The results demonstrate a simple method to purify and digest a recombinant IMP for analysis by mass spectrometry.     

High-Resolution Structure of a Membrane Protein Transferred from Amphipol to a Lipidic Mesophase

Amphipols (APols) have become important tools for the stabilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs solubilized in APols would be of high importance for structural biology. However, despite considerable efforts, it is still not clear whether MP/APol complexes can form well-ordered crystals suitable for X-ray crystallography. In the present work, we show that an APol-trapped MP can be crystallized in meso. Bacteriorhodopsin (BR) trapped by APol A8-35 was mixed with a lipidic mesophase, and crystallization was induced by adding a precipitant. The crystals diffract beyond 2 Å. The structure of BR was solved to 2 Å and found to be indistinguishable from previous structures obtained after transfer from detergent solutions. We suggest the proposed protocol of in meso crystallization to be generally applicable to APol-trapped MPs.