Proteomic profiling of secreted proteins from CHO cells using Surface-Enhanced Laser desorption ionization time-of-flight mass spectrometry

Chinese hamster ovary (CHO) cells are the most commonly used host cell line for the production of recombinant biopharmaceuticals. These biopharmaceuticals are typically secreted from CHO cells and purified from harvested cell culture media. The purpose of this study was to investigate changes in the secreted proteome of CHO cells over the various stages of the growth cycle using Surface Enhanced Laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS). Conditioned media samples were collected each day over a 6 day growth period from CHO-K1 cells grown in low serum (0.5% FBS) conditions in monolayer culture. Samples were profiled on a number of ProteinChip arrays with different chromatographic surfaces. From this study, 24 proteins were found to be differentially regulated at different phases of the growth cycle in CHO-K1 cells, when profiled on two chromatographic surfaces, Q10 (anionic) and IMAC30 (metal affinity) ProteinChip arrays.     

Proteomic profiling of erythrocyte proteins by proteolytic digestion chip and identification using two-dimensional electrospray ionization tandem mass spectrometry

Self-assembled monolayers (SAMs) on coinage metal provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition, and other interfacial phenomena. The bonding of enzyme to SAMs of alkanethiols onto gold surfaces is exploited to produce an enzyme chip. In this work, the attachment of trypsin to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water soluble N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as coupling agent. A two-dimensional liquid-phase separation scheme coupled with mass spectrometry is presented for proteomic analysis of erythrocyte proteins. The application of proteomics, particularly with reference to analysis of proteins, will be described. Surface analyses have revealed that the X-ray Photoelectron Spectroscopy (XPS) C1s and N1s core levels illustrate the immobilization of trypsin. These data are also in good agreement with Fourier Transformed Infrared Reflection-Attenuated Total Reflection (FTIR-ATR) spectra for the peaks at Amide I and Amide II. Using two-dimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nano-HPLC-ESI-MS/MS) system observations, analytical results have demonstrated the erythrocyte proteins digestion of the immobilized trypsin on the functionalized SAMs surface. For such surfaces, it also shows the enzyme digestion ability of the immobilized trypsin. The experiment results revealed the identification of 272 proteins from erythrocyte protein sample. The terminal groups of the SAMs structure can be further functionalized with biomolecules or antibodies to develop surface-base diagnostics, biosensors, or biomaterials.     

Analysis of mouse liver membrane proteins using multidimensional separations and tandem mass spectrometry

In the field of proteomic investigation, the analysis of membrane proteins still faces many technical challenges. A fundamental question in this puzzle is how to maintain a proper solvent environment to allow the hydrophobic proteins to remain solubilized. We propose that the denaturation of membrane proteins in a highly concentrated urea solution enables them to be ionized such that ionic exchange chromatography can be employed to separate them. The membrane proteins prepared from the mouse liver were dissolved in 6M guanidine hydrochloride, 20mM Tris-HCl, pH 9.0, and loaded onto a tandem chromatography apparatus coupled with Q-Sepharose FF and Sephacryl S-200HR. These columns were able to adsorb 97.87% of the membrane protein preparations. Using a linear NaCl (0-1.0M) gradient, the bound proteins were eluted out at 0.1-1.0M NaCl, and examined by SDS-PAGE. Furthermore the protein bands underwent excision and digestion with trypsin, followed by reverse-phase chromatography for the separation of the digested peptides and ionic-trap mass spectrometry for the identification of the proteins. From the SDS-PAGE gels, the overlap between proteins from neighboring bands was only 21.34%, indicating that the anionic-size exclusion coupling chromatography efficiently separated these membrane proteins. Of a total of 392 proteins identified, 306 were membrane proteins or membrane-associated proteins. Based on the calculation of hydrophobicity, the GRAVY scores of 83 proteins are greater than, or equal to, 0.00. Taking all of this evidence together, our results revealed that this approach is satisfactory for studies on the membrane proteome from the mouse liver.     

Proteomic profiling of human pleural effusion using two-dimensional nano liquid chromatography tandem mass spectrometry

Pleural effusion, an accumulation of pleural fluid, contains proteins originated from plasma filtrate and, especially when tissues are damaged, parenchyma interstitial spaces of lungs and/or other organs. This study details protein profiles in human pleural effusion from 43 lung adenocarcinoma patients by a two-dimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nano-HPLC-ESI-MS/MS) system. The experimental results revealed the identification of 1415 unique proteins from human pleural effusion. Among these 124 proteins identified with higher confidence levels, some proteins have not been reported in plasma and may represent proteins specifically present in pleural effusion. These proteins are valuable for mass identification of differentially expressed proteins involved in proteomics database and screening biomarker to further study in human lung adenocarcinoma. The significance of the use of proteomics analysis of human pleural fluid for the search of new lung cancer marker proteins, and for their simultaneous display and analysis in patients suffering from lung disorders has been examined.     

Proteomic characterization of wheat amyloplasts using identification of proteins by tandem mass spectrometry

We describe the initial characterization of the wheat amyloplast proteome, consisting of the identification and classification of 171 proteins. Whole amyloplasts and purified amyloplast membranes were prepared from wheat (Triticum aestivum). Protein extracts were examined by one-dimensional and two-dimensional electrophoresis, followed by high performance liquid chromatography-tandem mass spectrometry of separated proteins. Tandem mass spectrometry data of individual peptides was then searched by SEQUEST, using a database containing known protein sequences from both wheat and other homologous cereal crops. Using this approach we identified 108 proteins from whole amyloplasts and 63 proteins from purified amyloplast membranes. The majority of protein identifications were derived from protein sequences from cereal crops other than wheat, for which relatively little gene sequence data is available. The highest percentage of protein identifications obtained from any individual species was 46% of the total number of proteins identified, using sequence data found in our proprietary rice (Oryza sativa) genome database.     

FTICR mass spectrometry for qualitative and quantitative bioanalyses

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is playing an increasing role in the characterization of cellular systems owing to its capabilities for providing higher confidence of identification, increased dynamic range and sensitivity unmatched by other MS platforms. Particularly in proteomics, where global and quantitative approaches are essential, the attributes of FTICR-MS are poised to make significant contributions. Recent advances in the field that have particular importance for proteomic applications include the use of high-performance micro-capillary column separation techniques coupled to FTICR, as well as methods that improve protein identification, sensitivity, dynamic range and throughput.     

Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry

An approach to the systematic identification and quantification of the proteins contained in the microsomal fraction of cells is described. It consists of three steps: (1) preparation of microsomal fractions from cells or tissues representing different states; (2) covalent tagging of the proteins with isotope-coded affinity tag (ICAT) reagents followed by proteolysis of the combined labeled protein samples; and (3) isolation, identification, and quantification of the tagged peptides by multidimensional chromatography, automated tandem mass spectrometry, and computational analysis of the obtained data. The method was used to identify and determine the ratios of abundance of each of 491 proteins contained in the microsomal fractions of na?ve and in vitro- differentiated human myeloid leukemia (HL-60) cells. The method and the new software tools to support it are well suited to the large-scale, quantitative analysis of membrane proteins and other classes of proteins that have been refractory to standard proteomics technology.     

Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry

The objective of this study was to determine if liquid chromatography mass spectrometry (LC/MS) data of tryptic digests of proteins can be used for quantitation. In theory, the peak area of peptides should correlate to their concentration; hence, the peak areas of peptides from one protein should correlate to the concentration of that particular protein. To evaluate this hypothesis, different amounts of tryptic digests of myoglobin were analyzed by LC/MS in a wide range between 10 fmol and 100 pmol. The results show that the peak areas from liquid chromatography mass spectrometry correlate linearly to the concentration of the protein (r2 = 0.991). The method was further evaluated by adding two different concentrations of horse myoglobin to human serum. The results confirm that the quantitation method can also be used for quantitative profiling of proteins in complex mixtures such as human sera. Expected and calculated protein ratios differ by no more than 16%. We describe a new method combining protein identification with accurate profiling of individual proteins. This approach should provide a widely applicable means to compare global protein expression in biological samples.     

Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.

Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray-ionization mass spectrometry (ESI-MS) of intact intrinsic membrane proteins purified using reverse-phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the D1 and D2 reaction-center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI-MS study of a G-protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post-translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI-MS experiments.     

Proteomic profiling and protein identification by MALDI-TOF mass spectrometry in unsequenced parasitic nematodes

Lack of genomic sequence data and the relatively high cost of tandem mass spectrometry have hampered proteomic investigations into helminths, such as resolving the mechanism underpinning globally reported anthelmintic resistance. Whilst detailed mechanisms of resistance remain unknown for the majority of drug-parasite interactions, gene mutations and changes in gene and protein expression are proposed key aspects of resistance. Comparative proteomic analysis of drug-resistant and -susceptible nematodes may reveal protein profiles reflecting drug-related phenotypes. Using the gastro-intestinal nematode, Haemonchus contortus as case study, we report the application of freely available expressed sequence tag (EST) datasets to support proteomic studies in unsequenced nematodes. EST datasets were translated to theoretical protein sequences to generate a searchable database. In conjunction with matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), Peptide Mass Fingerprint (PMF) searching of databases enabled a cost-effective protein identification strategy. The effectiveness of this approach was verified in comparison with MS/MS de novo sequencing with searching of the same EST protein database and subsequent searches of the NCBInr protein database using the Basic Local Alignment Search Tool (BLAST) to provide protein annotation. Of 100 proteins from 2-DE gel spots, 62 were identified by MALDI-TOF-MS and PMF searching of the EST database. Twenty randomly selected spots were analysed by electrospray MS/MS and MASCOT Ion Searches of the same database. The resulting sequences were subjected to BLAST searches of the NCBI protein database to provide annotation of the proteins and confirm concordance in protein identity from both approaches. Further confirmation of protein identifications from the MS/MS data were obtained by de novo sequencing of peptides, followed by FASTS algorithm searches of the EST putative protein database. This study demonstrates the cost-effective use of available EST databases and inexpensive, accessible MALDI-TOF MS in conjunction with PMF for reliable protein identification in unsequenced organisms.     

Chemokine-glycosaminoglycan binding: specificity for CCR2 ligand binding to highly sulfated oligosaccharides using FTICR mass spectrometry

Glycosaminoglycans (GAGs) have recently been demonstrated to be required for the in vivo activity of several chemokines. Minimally, the interaction is thought to provide a mechanism for retention at the site of secretion and the formation of chemokine gradients that provide directional cues for receptor bearing cells, particularly in the presence of shear forces. Thus, a key issue will be to determine the sequence and structure of the GAGs that bind to specific chemokines. Herein, we describe a mass spectrometry assay that was developed to detect protein-oligosaccharide noncovalent complexes, in this case chemokine-GAG interactions, and to select for high affinity GAGs. The process is facilitated by the ability of electrospray ionization to transfer the intact noncovalent complexes from solution into the gas phase. The elemental composition as well as the binding stoichiometry can be calculated from the mass of the complex. Ligands of the chemokine receptor, CCR2 (MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, MCP-4/CCL13, and Eotaxin/CCL11), and the CCR10 ligand CTACK/CCL27 were screened against a small, highly sulfated, heparin oligosaccharide library with limited structural variation. The results revealed heparin octasaccharides with 11 and 12 sulfates as binders. Oligomerization of some chemokines was observed upon GAG binding, whereas in other instances only the monomeric noncovalent complex was identified. The results indicate that, in contrast to the apparent redundancy in the chemokine system, where several chemokines bind and activate the same receptor, these chemokines could be differentiated into two groups based on the stoichiometry of their complexes with the heparin oligosaccharides.     

Proteomic analysis and discovery using affinity proteomics and mass spectrometry

Antibody-based microarrays are a rapidly evolving affinity-proteomic methodology that recently has shown great promise in clinical applications. The resolution of these proteomic analyses is, however, directly related to the number of data-points, i.e. antibodies, included on the array. Currently, this is a key bottleneck because of limited availability of numerous highly characterized antibodies. Here, we present a conceptually new method, denoted global proteome survey, opening up the possibility to probe any proteome in a species-independent manner while still using a limited set of antibodies. We use context-independent-motif-specific antibodies directed against short amino acid motifs, where each motif is present in up to a few hundred different proteins. First, the digested proteome is exposed to these antibodies, whereby motif-containing peptides are enriched, which then are detected and identified by mass spectrometry. In this study, we profiled extracts from human colon tissue, yeast cells lysate, and mouse liver tissue to demonstrate proof-of-concept.     

Characterisation of intact microorganisms using electrospray ionisation mass spectrometry

We report the first application of electrospray ionisation mass spectrometry (ESI-MS) for the reproducible characterisation of strains of intact Gram-negative and Gram-positive bacteria. Electrospray ionisation was performed in both the positive and negative ion modes and the spectra obtained from Escherichia coli and Bacillus cereus were very information rich. Several of the observed negative mass ion fragments from E. coli could be assigned to specific fragmentation from bacterial phospholipids.Cluster analyses of these spectra showed that ESI-MS could be used to discriminate between these microorganisms to below species level. Therefore we conclude that ESI-MS constitutes a powerful approach to the characterisation and speciation of intact microorganisms.     

Comparative profiling of human saliva by intact protein LC/ESI-TOF mass spectrometry

Human saliva is finding increasing interest for proteomic and biomarker-discovery studies, due to the ease of collection and potential for simpler processing workflows compared to serum or plasma. However, it is known that salivary protein composition can vary with physiological and environmental factors. In this work, we have examined intra- and inter-person variability of saliva protein composition using an LC/MS methodology to profile low molecular weight human salivary proteins. Whole saliva was analyzed from four individuals over three consecutive days. Additional samples were used to determine baseline analytical and sample processing variation and to identify phosphoproteins. Individuals were observed to have a similar salivary protein pattern over multiple days, although the expression levels of particular proteins were variable. Significant differences in protein profiles were observed between subjects, allowing for delineation of individuals based on their protein profile. Comparison with alkaline phosphatase treated saliva revealed that several identified proteins were singly, doubly, or triply phosphorylated.     

Proteomic analysis of synaptosomes using isotope-coded affinity tags and mass spectrometry

Synaptosomes are isolated synapses produced by subcellular fractionation of brain tissue. They contain the complete presynaptic terminal, including mitochondria and synaptic vesicles, and portions of the postsynaptic side, including the postsynaptic membrane and the postsynaptic density (PSyD). A proteomic characterisation of synaptosomes isolated from mouse brain was performed employing the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS/MS). After isotopic labelling and tryptic digestion, peptides were fractionated by cation exchange chromatography and cysteine-containing peptides were isolated by affinity chromatography. The peptides were identified by microcapillary liquid chromatography-electrospray ionisation MS/MS (muLC-ESI MS/MS). In two experiments, peptides representing a total of 1131 database entries were identified. They are involved in different presynaptic and postsynaptic functions, including synaptic vesicle exocytosis for neurotransmitter release, vesicle endocytosis for synaptic vesicle recycling, as well as postsynaptic receptors and proteins constituting the PSyD. Moreover, a large number of soluble and membrane-bound molecules serving functions in synaptic signal transduction and metabolism were detected. The results provide an inventory of the synaptic proteome and confirm the suitability of the ICAT method for the assessment of synaptic structure, function and plasticity.     

Proteomic profiling of hempseed proteins from Cheungsam

Proteomic profiling of hempseed proteins from a non-drug type of industrial hemp (Cannabis sativa L.), Cheungsam, was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 1102 protein spots were resolved on pH 3-10 immobilized pH gradient strips, and 168 unique protein spots were identified. The proteins were categorized based on function, including involvement in energy regulation (23%), metabolism (18%), stress response (16%), unclassified (12%), cytoskeleton (11%), binding function (5%), and protein synthesis (3%). These proteins might have important biological functions in hempseed, such as germination, storage, or development.     

Direct profiling and imaging of plant metabolites in intact tissues by using colloidal graphite-assisted laser desorption ionization mass spectrometry

Laser desorption/ionization (LDI)-based imaging mass spectrometry (MS) has been applied to several biological systems to obtain information about both the identities of the major chemical species and their localization. Colloidal graphite-assisted LDI (GALDI) MS imaging was introduced for the imaging of small molecules such as phospholipids, cerebrosides, oligosaccharides, flavonoids, and other secondary metabolites with high spatial homogeneity due to finely dispersed particles. Mass profiles and images of Arabidopsis thaliana have been recorded directly from various plant surfaces and cross sections. The main targeted metabolites were flavonoids and cuticular waxes, both of which are important in many aspects of functional genomics, proteomics, and metabolomics. The mass spectral profiles revealed tissue-specific accumulation of flavonoids in flowers and petals. In addition, many other location-specific ions were observed. The location and the degree of light-induced accumulation of flavonoids in stem sections were successfully probed by GALDI MS.     

Proteomic profiling of hempseed proteins from Cheungsam

Proteomic profiling of hempseed proteins from a non-drug type of industrial hemp (Cannabis sativa L.), Cheungsam, was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 1102 protein spots were resolved on pH 3-10 immobilized pH gradient strips, and 168 unique protein spots were identified. The proteins were categorized based on function, including involvement in energy regulation (23%), metabolism (18%), stress response (16%), unclassified (12%), cytoskeleton (11%), binding function (5%), and protein synthesis (3%). These proteins might have important biological functions in hempseed, such as germination, storage, or development.