High recovery HPLC separation of lipid rafts for membrane proteome analysis

Proteomic analysis of complex samples can be facilitated by protein fractionation prior to enzymatic or chemical fragmentation combined with MS-based identification of peptides. Although aqueous soluble protein fractionation by liquid chromatography is relatively straightforward, membrane protein separations have a variety of technical challenges. Reversed-phase high performance liquid chromatography (RP-HPLC) separations of membrane proteins often exhibit poor recovery and bandwidths, and generally require extensive pretreatment to remove lipids and other membrane components. Human brain tissue lipid raft protein preparations have been used as a model system to develop RP-HPLC conditions that are effective for protein fractionation, and are compatible with downstream proteomic analytical workflows. By the use of an appropriate RP column material and operational conditions, human brain membrane raft proteins were successfully resolved by RP-HPLC and some of the protein components, including specific integral membrane proteins, identified by downstream SDS-PAGE combined with in-gel digestion, or in-solution digestion and LC-MS/MS analysis of tryptic fragments. Using the described method, total protein recovery was high, and the repeatability of the separation maintained after repeated injections of membrane raft preparations.     

Multidimensional chromatography: a powerful tool for the analysis of membrane proteins in mouse brain

Understanding the function of membrane proteins is of fundamental importance due to their crucial roles in many cellular processes and their direct association with human disorders. However, their analysis poses a special challenge, largely due to their highly amphipathic nature. Until recently, analyses of proteomic samples mainly were performed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), due to the unprecedented separation power of the technique. However, in conventional 2D-PAGE membrane proteins are generally underrepresented due to their tendency to precipitate during isoelectric focusing and their inefficient transfer from the first to the second dimension. As a consequence, several other separation techniques, primarily based on liquid chromatography (LC), have been employed for analysis of this group of proteins. In the present study, different LC-based methods were compared for the analysis of crude protein extracts. One- and two-dimensional high-performance liquid chromatographic (1D- and 2D-HPLC) separations of brain protein tryptic digests with a predicted concentration range of up to 5 orders of magnitude were found to be insufficient, thus making a preceding fractionation step necessary. An additional protein separation step was introduced and a 3D-PAGE-HPLC analysis was performed. The results of these experiments are compared with results of 2D-PAGE/matrix-assisted laser desorption ionization mass spectrometric (MALDI MS) analyses of the same samples. Features, challenges, advantages, and disadvantages of the respective systems are discussed. The brain (mouse and human) was chosen as the analyzed tissue as it is of high interest in medical and pharmaceutical research into neurological diseases such as multiple sclerosis, stroke, Alzheimer's disease, and Parkinson's disease. The study is part of our ongoing research aimed at identifying new biomarkers for neurodegenerative diseases.     

Application of displacement chromatography for the analysis of a lipid raft proteome

Defining membrane proteomes is fundamental to understand the role of membrane proteins in biological processes and to find new targets for drug development. Usually multidimensional chromatography using step or gradient elution is applied for the separation of tryptic peptides of membrane proteins prior to their mass spectrometric analysis. Displacement chromatography (DC) offers several advantages that are helpful for proteome analysis. However, DC has so far been applied for proteomic investigations only in few cases. In this study we therefore applied DC in a multidimensional LC–MS approach for the separation and identification of membrane proteins located in cholesterol-enriched membrane microdomains (lipid rafts) obtained from rat kidney by density gradient centrifugation. The tryptic peptides were separated on a cation-exchange column in the displacement mode with spermine used as displacer. Fractions obtained from DC were analyzed using an HPLC-chip system coupled to an electrospray-ionization ion-trap mass spectrometer. This procedure yielded more than 400 highly significant peptide spectrum matches and led to the identification of more than 140 reliable protein hits within an established rat kidney lipid raft proteome. The majority of identified proteins were membrane proteins. In sum, our results demonstrate that DC is a suitable alternative to gradient elution separations for the identification of proteins via a multidimensional LC–MS approach.     

Improvement of hydrophobic integral membrane protein identification by mild performic acid oxidation-assisted digestion

Integral membrane proteins (IMPs) are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of IMPs make them difficult to analyze. In proteomic analyses, hydrophobic peptides including transmembrane domains are often underrepresented, and this reduces the sequence coverage and reliability of the identified IMPs. Here we report a new strategy, mild performic acid oxidation treatment (mPAOT), for improvement of IMP identification. In the mPAOT strategy, the hydrophobicity of IMPs is significantly decreased by oxidizing their methionine and cysteine residues with performic acid, thereby improving the solubility and enzymolysis of these proteins. The application of the mPAOT strategy to the analysis of IMPs from human nasopharyngeal carcinoma CNE1 cell line demonstrated that many IMPs, including those with high hydrophobicity, could be reliably identified.     

Proteomic analyses using an accurate mass and time tag strategy

An accurate mass and time (AMT) tag approach for proteomic analyses has been developed over the past several years to facilitate comprehensive high-throughput proteomic measurements. An AMT tag database for an organism, tissue, or cell line is established by initially performing standard shotgun proteomic analysis and, most importantly, by validating peptide identifications using the mass measurement accuracy of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) and liquid chromatography (LC) elution time constraint. Creation of an AMT tag database largely obviates the need for subsequent MS/MS analyses, and thus facilitates high-throughput analyses. The strength of this technology resides in the ability to achieve highly efficient and reproducible one-dimensional reversed-phased LC separations in conjunction with highly accurate mass measurements using FTICR MS. Recent improvements allow for the analysis of as little as picrogram amounts of proteome samples by minimizing sample handling and maximizing peptide recovery. The nanoproteomics platform has also demonstrated the ability to detect >10(6) differences in protein abundances and identify more abundant proteins from subpicogram amounts of samples. The AMT tag approach is poised to become a new standard technique for the in-depth and high-throughput analysis of complex organisms and clinical samples, with the potential to extend the analysis to a single mammalian cell.     

Treponema pallidum rare outer membrane proteins: analysis of mobility by freeze-fracture electron microscopy.

Freeze-fracture and deep-etch electron microscopy were used to investigate the molecular architecture of the Treponema pallidum outer membrane (OM). Freeze-fracture electron microscopy of treponemes freshly harvested from rabbit testes revealed that the intramembranous particles (IMPs) in both the concave and convex OM leaflets were distributed into alternating areas of relatively high and low particle density; in many OM fractures, IMPs formed rows that ran either parallel to or obliquely across the fracture faces. Statistical analysis (runs test) confirmed that the IMPs were nonrandomly distributed in both OM leaflets. Examination of deep-etched specimens revealed that the particles observed in freeze-fractured OMs also were surface exposed. Combined analysis of deep-etched and cross-fractured treponemes revealed that the OM particles were located in regions of the OM away from the endoflagella and closely apposed to the cytoplasmic membrane-peptidoglycan complex. When treponemes were incubated for extended periods with heat-inactivated immune rabbit syphilitic serum, no alteration in the distribution of OM IMPs was detected. In further experiments, approximately 1:1 mixtures of T. pallidum and Escherichia coli or separate suspensions of the nonpathogenic Treponema phagedenis biotype Reiter were fixed at 34 degrees C or after cooling to 0 degree C (to induce lateral phase separations that would aggregate IMPs). Only particles in the T. pallidum OM failed to aggregate in cells fixed at the lower temperature. The combined data suggest that the mobility of T. pallidum rare OM proteins is limited, perhaps as a result of interactions between their periplasmic domains and components of the peptidoglycan-cytoplasmic membrane complex.     

A combination of immobilised pH gradients improves membrane proteomics

Membrane protein analyses have been notoriously difficult due to hydrophobicity and the general low abundance of these proteins compared to their soluble cytosolic counterparts. Shotgun proteomics has become the preferred method for analyses of membrane proteins, in particular the recent development of peptide immobilized pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two-dimensional shotgun proteomics. Recently, peptide IPG-IEF has been shown to be a valuable shotgun proteomics technique through the use of acidic narrow range IPG strips, which demonstrated that small acidic p I increments are rich in peptides. In this study, we assess the utility of both broad range (BR) (p I 3-10) and narrow range (NR) (p I 3.4-4.9) IPG strips for rat liver membrane protein analyses. Furthermore, the use of these IPG strips was evaluated using label-free quantitation to demonstrate that the identification of a subset of proteins can be improved using NR IPG strips. NR IPG strips provided 2603 protein assignments on average (with 826 integral membrane proteins (IMPs)) compared to BR IPG strips, which provided 2021 protein assignments on average (with 712 IMPs). Nonredundant protein analysis demonstrated that in total from all experiments, 4195 proteins (with 1301 IMPs) could be identified with 1428 of these proteins unique to NR IPG strips with only 636 from BR IPG strips. With the use of label-free quantitation methods, 1659 proteins were used for quantitative comparison of which 319 demonstrated statistically significant increases in normalized spectral abundance factors (NSAF) in NR IPG strips compared to 364 in BR IPG strips. In particular, a selection of six highly hydrophobic transmembrane proteins was observed to increase in NSAF using NR IPG strips. These results provide evidence for the use of alternative pH gradients in combination to improve the shotgun proteomic analysis of the membrane proteome.     

Liquid chromatography MALDI MS/MS for membrane proteome analysis

Membrane proteins play critical roles in many biological functions and are often the molecular targets for drug discovery. However, their analysis presents a special challenge largely due to their highly hydrophobic nature. We present a surfactant-aided shotgun proteomics approach for membrane proteome analysis. In this approach, membrane proteins were solubilized and digested in the presence of SDS followed by newly developed auto-offline liquid chromatography/matrix-assisted laser desorption ionization (LC/MALDI) tandem MS analysis. Because of high tolerance of MALDI to SDS, one-dimensional (1D) LC separation can be combined with MALDI for direct analysis of protein digests containing SDS, without the need for extensive sample cleanup. In addition, the heated droplet interface used in LC/MALDI can work with high flow LC separations, allowing a relatively large amount of protein digest to be used for 1D LC/MALDI which facilitates the detection of low abundance proteins. The proteome identification results obtained by LC/MALDI are compared to the gel electrophoresis/MS method as well as the shotgun proteomics method using 2D LC/electrospray ionization MS. It is demonstrated that, while LC/MALDI provides more extensive proteome coverage compared to the other two methods, these three methods are complementary to each other and a combination of these methods should provide a more comprehensive membrane proteome analysis.     

Solubilization of native integral membrane proteins in aqueous buffer by noncovalent chelation with monomethoxy poly(ethylene glycol) (mPEG) polymers

Highly hydrophobic integral membrane proteins (IMPs)are typically purified in excess detergent media, often resulting in rapid inactivation and denaturation of the protein. One promising approach to solve this problem is to couple hydrophilic polymers, such as monomethoxypolyethylene glycol (mPEG) to IMPs under mild conditions in place of detergents. However, the broad application of this approach is hampered by poor reaction efficiencies, low tolerance of detergent stabilized membrane proteins to reaction conditions, and a lack of proper site-specific reversible approaches. Here, we have developed a straightforward, efficient, and mild approach to site-specific noncovalent binding of long-chain polymers to recombinant IMPs. This method uses the hexa-histidine tag (His-Tag) often used for purification of recombinant proteins as an attachment site for mPEGs. Solubility studies performed using five different IMPs confirmed that all tested mPEG-bound IMPs were completely soluble and stable in detergent free aqueous buffer compared to their precipitated native proteins under the identical circumstances. Activity assays and circular dichroism (CD) spectroscopy confirmed the structural integrity of modified IMPs.     

Proteome profile of cytosolic component of zebrafish liver generated by LC-ESI MS/MS combined with trypsin digestion and microwave-assisted acid hydrolysis

The zebrafish genome has recently been sequenced and annotated allowing for high-throughput proteomic analysis. Here, we report for the first time a proteomic subset of zebrafish liver, an important organ for metabolizing toxins. Using a newly developed analytical procedure, we have identified 1204 proteins from the cytosolic component of a zebrafish liver tissue sample. Our methods involve cell-compartment fractionation of liver tissue samples, four levels of protein digestion, and off-line two-dimensional liquid chromatography (2-D LC) separations of resultant peptides. Proteins are identified using an electrospray ionization quadrupole time-of-flight tandem mass spectrometer (ESI-QTOF MS/MS), which provides high-resolution and high-accuracy mass measurement of peptide ions and their fragment ions. We demonstrate that greater proteome coverage can be achieved by combining the results obtained from four methods of protein digestion: three tryptic digests (one in buffer, one in methanol, and another in SDS), and a microwave-assisted acid hydrolysate of the protein extracts. Identified proteins--which included several groups of established protein biomarkers--were functionally classified. We discuss the functions and implications of these biomarkers within the context of zebrafish toxicology.     

Protein based microarrays: A tool for probing the proteome of cancer cells and tissues

A novel proteomic approach for probing cell and tissue proteome, which combines liquid phase protein separations with microarray technology has been developed. Proteins in cell and tissue lysates or in cellular subfractions are separated using any one of a number of separation modes which may consist of ion exchange liquid chromatography (LC), reverse phase LC, carrier ampholyte based separations, e.g. the use of Rotofor, affinity based separations, or gel based separations. Each first-dimension fraction obtained using one separation mode can be further resolved using one or more of the other separation modes to yield either purified protein in solution or liquid fractions with substantially reduced protein complexity. The advantage of a liquid based separation system is that proteins in hundreds of individual fractions can be arrayed directly and used as targets for a variety of probes. Constituent proteins in reactive fractions are identified by mass spectrometry and may be further resolved to determine the nature of the reactive protein(s). We present in this report initial data based on microarray analysis of individual Rotofor fractions obtained from lung adenocarcinoma cell line A549 lysates which have been probed with antibodies against specific proteins.     

Detection of plasma membrane cholesterol by filipin during microvillogenesis and ciliogenesis in quail oviduct

Using filipin as a probe for the presence of membrane cholesterol, the evolution of cholesterol distribution in the apical plasma membrane was studied during estrogen-induced ciliogenesis in quail oviduct and compared with the distribution of intramembrane particles (IMPs). Ciliary growth is preceded by the first step of microvillus differentiation. Microvilli emerge in membrane domains rich in IMPs and devoid of filipin-cholesterol (f-c) complexes. However growing microvillus membrane shows f-c complexes. During ciliary growth, microvilli lengthen from 0.5 to 2 microns, indicating that the microvillar membrane is not a membrane reservoir for ciliogenesis. During ciliary growth, the characteristic ciliary necklace IMP rows appear progressively at the base of cilia. The first IMP row is organized in a membrane circlet lacking of f-c complexes, whereas the new shaft membrane in the middle of the circlet exhibits numerous complexes. These two different domains of the cilia keep their specificity during ciliary growth. Only the ciliary tip shows fewer complexes than the shaft membrane. The apical membrane of differentiated ciliated cells is thus composed of various domains, the ciliary shaft full of f-c complexes and poor in IMPs, the ciliary necklace is devoid of f-c complexes and rich in IMPs, the microvilli membrane is rich in both IMPs and f-c complexes, and the interciliary membrane is poor in both f-c complexes and IMPs, whereas the undifferentiated cells exhibit an apical membrane in which f-c complexes and IMPs are distributed homogeneously.     

Proteomics for Protein Expression Profiling in Neuroscience

As the technology of proteomics moves from a theoretical approach to a practical reality, neuroscientists will have to determine the most appropriate applications for this technology. Neuroscientists will have to surmount difficulties particular to their research, such as limited sample amounts, heterogeneous cellular compositions in samples, and the fact that many proteins of interest are rare, hydrophobic proteins. This review examines protein isolation and protein fractionation and separation using two-dimensional electrophoresis (2-DE) and mass spectrometry proteomic methods. Methods for quantifying relative protein expression between samples (e.g., 2-DIGE, and ICAT) are also described. The coverage of the proteome, ability to detect membrane proteins, resource requirements, and quantitative reliability of different approaches is also discussed. Although there are many challenges in proteomic neuroscience, this field promises many rewards in the future.     

Shotgun proteomic analysis of cerebrospinal fluid using off-gel electrophoresis as the first-dimension separation

Shotgun proteomic analysis usually employs multidimensional separations with the first dimension most commonly being strong cation exchange (SCX) liquid chromatography (LC). SCX-LC is necessarily a serial process for preparation of multiple samples. Here, we apply a newly available tool, off-gel electrophoresis (OGE), for first-dimension separation of peptide mixtures from digests of cerebrospinal fluid (CSF), a complex and low total protein-containing sample. OGE first-dimension fractionation enabled identification of a total of 156 unique proteins compared to 115 identified in previous work using first-dimension SCX fractionation. OGE can be used to process multiple samples unattended with easy retrieval of the separated fractions. Thus, shotgun analysis using OGE as the first-dimension separation offers a significant advantage both in terms of sample throughput as well as increased numbers of identified proteins.     

Disruption and reformation of the acetylcholine receptor clusters of cultured rat myotubes occur in two distinct stages

We have examined the redistribution of acetylcholine receptor (AChR) intramembrane particles (IMPs) when AChR clusters of cultured rat myotubes are experimentally disrupted and allowed to reform. In control myotubes, the AChR IMPs are evenly distributed within the AChR domains of cluster membrane. Shortly after addition of azide to disrupt clusters, IMPs become unevenly scattered, with some microaggregation. After longer treatment, IMPs are depleted from AChR domains with no further change in IMP distribution. Contact domains of clusters are relatively poor in IMPs both before and after cluster dispersal. Upon visualization with fluorescent alpha-bungarotoxin, some AChR in azide-treated samples appear as small, bright spots. These spots do not correspond to microaggregates seen in freeze-fracture replicas, and probably represent receptors that have been internalized. The internalization rate is insufficient to account completely for the loss of IMPs from clusters, however. During reformation of AChR clusters upon removal of azide, IMP concentration in receptor domains increases. At early stages of reformation, IMPs appear in small groups containing compact microaggregates. At later times, AChR domains enlarge and IMPs within them assume the evenly spaced distribution characteristic of control clusters. These observations suggest that the disruption of clusters is accompanied by mobilization of AChR from a fixed array, allowing AChR IMPs to diffuse away from the clusters, to form microaggregates, and to become internalized. Cluster reformation appears to be the reverse of this process. Our results are thus consistent with a two-step model for AChR clustering, in which the concentration of IMPs into a small membrane region precedes their rearrangement into evenly spaced sites.     

Characterization of the consequences of YidC depletion on the inner membrane proteome of E. coli using 2D blue native/SDS-PAGE

In the bacterium Escherichia coli, the essential inner membrane protein (IMP) YidC assists in the biogenesis of IMPs and IMP complexes. Our current ideas about the function of YidC are based on targeted approaches using only a handful of model IMPs. Proteome-wide approaches are required to further our understanding of the significance of YidC and to find new YidC substrates. Here, using two-dimensional blue native/SDS-PAGE methodology that is suitable for comparative analysis, we have characterized the consequences of YidC depletion for the steady-state levels and oligomeric state of the constituents of the inner membrane proteome. Our analysis showed that (i) YidC depletion reduces the levels of a variety of complexes without changing their composition, (ii) the levels of IMPs containing only soluble domains smaller than 100 amino acids are likely to be reduced upon YidC depletion, whereas the levels of IMPs with at least one soluble domain larger than 100 amino acids do not, and (iii) the levels of a number of proteins with established or putative chaperone activity (HflC, HflK, PpiD, OppA, GroEL and DnaK) are strongly increased in the inner membrane fraction upon YidC depletion. In the absence of YidC, these proteins may assist the folding of sizeable soluble domains of IMPs, thereby supporting their folding and oligomeric assembly. In conclusion, our analysis identifies many new IMPs/IMP complexes that depend on YidC for their biogenesis, responses that accompany depletion of YidC and an IMP characteristic that is associated with YidC dependence.     

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

Integral membrane proteins (IMPs) control countless fundamental biological processes and constitute the majority of drug targets. For this reason, uncovering their molecular mechanism of action has long been an intense field of research. They are, however, notoriously difficult to work with, mainly due to their localization within the heterogeneous of environment of the biological membrane and the instability once extracted from the lipid bilayer. High‐resolution structures have unveiled many mechanistic aspects of IMPs but also revealed that the elucidation of static pictures has limitations. Hydrogen–deuterium exchange coupled to mass spectrometry (HDX‐MS) has recently emerged as a powerful biophysical tool for interrogating the conformational dynamics of proteins and their interactions with ligands. Its versatility has proven particularly useful to reveal mechanistic aspects of challenging classes of proteins such as IMPs. This review recapitulates the accomplishments of HDX‐MS as it has matured into an essential tool for membrane protein structural biologists.     

Strategies for shotgun identification of integral membrane proteins by tandem mass spectrometry

Integral membrane proteins (IMPs) are difficult to identify, mainly for two reasons: the hydrophobicity of IMPs and their low abundance. Sample preparation is a key component in the large-scale identification of IMPs. In this review, we survey strategies for shotgun identification of IMPs by MS/MS. We will discuss enrichment, solubilization, separation, and digestion of IMPs, and data analysis for membrane proteomics.