Analysis of Human Plasma Proteome by 2DE‐ and 2D nanoLC‐Based Mass Spectrometry

Abstract

We compared the 2DE coupled to MALDI‐TOF‐MS and ESI‐MS/MS analysis (2DE‐MS) and the on‐line 2D nanoLC, followed by nanoESI‐MS/MS analysis (2DLC‐MS), for the separation and identification of proteins in high abundance protein‐depleted human plasma. Identification of proteins in the plasma by the two methods demonstrated that the majority of the identified protein set was unique to each method. Therefore, if a comprehensive coverage of the proteome identification is desired, it is ideal to apply both methods. The 2DE‐MS method is amenable to protein spot‐based quantitation, whereas the 2DLC‐MS method may provide an advantage of the high throughput application.     

An improved method of sample preparation on AnchorChip targets for MALDI-MS and MS/MS and its application in the liver proteome project

An improved method for sample preparation for MALDI-MS and MS/MS using AnchorChip targets is presented. The method, termed the SMW method (sample, matrix wash), results in better sensitivity for peptide mass fingerprinting as well as for sequencing by MS/MS than previously published methods. The method allows up-concentration and desalting directly on the mass spectrometric target and should be amenable for automation. A draw back caused by extensive oxidation of methionine and tryptophan in the SMW method can be alleviated by the addition of n-octyl glucopyranoside and DTT to the sample solution. The method was validated for protein identification from a 2-DE based liver proteome study. The SMW method resulted in identification of many more proteins and in most cases with a better score than the previously published methods.     

Comparison of 2-D LC and 3-D LC with post- and pre-tryptic-digestion SEC fractionation for proteome analysis of normal human liver tissue

The current "shotgun" proteomic analysis, strong cation exchange-RPLC-MS/MS system, is a widely used method for proteome research. Currently, it is not suitable for complicated protein sample analysis, like mammal tissues or cells. To increase the protein identification confidence and number, an additional separation dimension for sample fractionation is necessary to be coupled prior to current multi-dimensional protein identification technology (MudPIT). In this work, SEC was elaborately selected and applied for sample prefractionation in consideration of its non-bias against sample and variety of choice of mobile phases. The analysis of the global lysate of normal human liver tissue sample provided by the China Human Liver Proteome Project, were performed to compare the proteome coverage, sequence coverage (peptide per protein identification) and protein identification efficiency in MudPIT, 3-D LC-MS/MS identification strategy with preproteolytic and postproteolytic fractionation. It was demonstrated that 3-D LC-MS/MS utilizing protein level fractionation was the most effective method. A MASCOT search using the MS/MS results acquired by QSTAR(XL) identified 1622 proteins from 3-D LC-MS/MS identification approaches. A primary analysis on molecular weight, pI and grand average hydrophobicity value distribution of the identified proteins in different approaches was made to further evaluate the 3-D LC-MS/MS analysis strategy.     

Molecular mass sorting of proteome using hollow fiber flow field-flow fractionation for proteomics

Hollow fiber flow field-flow fractionation (HF FlFFF) has been demonstrated as a tool for pre-fractionating proteomes by differences in molecular mass (Mr), where the resulting protein fractions are subsequently digested and analyzed by shotgun proteomics using two-dimensional liquid chromatography-electrospray ionization-tandem mass spectrometry (2D-LC-ESI-MS/MS). HF FlFFF is a separation device capable of fractionating proteins or cells by hydrodynamic radius, and protein fraction can be readily collected as intact conditions in aqueous buffer solutions. In this study, HF FlFFF was applied to fractionate the proteome of Corynebacterium glutamicum, a well known soil bacterium that has been widely used in bioindustry due to its remarkable ability to secrete high amounts of glutamic acid. The collected HF FlFFF fractions of different MW intervals were enzymatically digested for protein identification by 2D-LC-ESI-MS/MS. Experiments showed improvements in protein identification when HF FlFFF pre-fractionation was applied, due to decreases in the ionization suppression effect and the MS exclusion effect by spectral congestion. Pre-fractionation of C. glutamicum proteome allowed us to find 90 additional proteins by 2D-LC-ESI-MS/MS that were not found by a direct shotgun analysis without pre-fractionation. A total of 415 proteins were found overall with 203 proteins commonly found from experiments with and without pre-fractionation.     

1-DE MS and 2-D LC-MS analysis of the mouse bronchoalveolar lavage proteome

Bronchoalveolar lavage fluid (BALF) is a complex mixture of proteins, which represents a unique clinically useful sampling of the lower respiratory tract. Many proteomic technologies can be used to characterize complex biological mixtures; however, it is not yet clear which technology(s) provide more information regarding the number of proteins identified and sequence coverage. In this study, we initially compared two common proteomic approaches, 2-D LC microESI MS/MS and 1-DE followed by gel slice digestion, peptide extraction and peptide identification by MS in characterization of the mouse BALF proteome; secondly, we identified 297 unique proteins from the mouse BALF proteome, greatly expanded the BALF proteome by about threefold regardless of species.     

Evaluation of three‐dimensional gel electrophoresis to improve quantitative profiling of complex proteomes

Two‐dimensional remains one of the main experimental approaches in proteome analysis. However, comigration of protein leads to several limitations: lack of accuracy in protein identification, impaired comparative quantification, and PTM detection. We have optimized a third additional step of in‐gel separation to alleviate comigration associated drawbacks. Spot resolution is strikingly improved following this simple and rapid method and the positive impact on protein and peptide identification from MS/MS data, on the analysis of relative changes in protein abundance, and on the detection of PTM is described.     

Prefractionation of proteome by liquid isoelectric focusing prior to two-dimensional liquid chromatography mass spectrometric identification

Due to the complexity of proteomes, developing methods of sample fractionation, separation, concentration, and detection have become urgent to the identification of large numbers of proteins, as well as the acquisition of those proteins in low abundance. In this work, liquid isoelectric focusing (LIEF) combined with 2D-LC-MS/MS was applied to the proteome of Saccharomyces cerevisiae. This yielded a total of 1795 proteins that were detected and identified by 30 fractions of protein prefractionation. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis without protein fractionation. LIEF-2D-LC-MS/MS also produced improved resolution of low-abundance proteins. Furthermore, we analyzed the characteristics of proteins obtained by LIEF-2D-LC-MS/MS. 1103 proteins with CAI under 0.2 were identified, allowing us to specifically obtain detailed biochemical information on these kind proteins. It was observed that LIEF-2D-LC-MS/MS is useful for large-scale proteome analysis and may be specifically applied to systems with wide dynamic ranges.     

Proteomic profiling of hepatocellular carcinoma in Chinese cohort reveals heat-shock proteins (Hsp27, Hsp70, GRP78) up-regulation and their associated prognostic values

To facilitate the identification of candidate molecular biomarkers that are linked to the pathogenesis of hepatocellular carcinoma (HCC), we investigated protein-expression profiles of 146 tissue specimens including 67 pairs of tumors and adjacent non-tumors resected from HCC patients as well as 12 normal livers by 2-DE. Among the 1800 spots displayed in the liver proteome, a total of 90 protein species were found to be significantly different between the three groups (P < 0.05). Three of the top candidate markers up-regulated in HCC, with high receiver operating characteristic (ROC) curves, were identified by MS/MS analysis and belonged to the chaperone members: heat-shock protein (Hsp)27, Hsp70 and glucose-regulated protein (GRP)78. Over-expression of these chaperone proteins in HCC tissues was confirmed by Western blotting and immunohistochemistry. In correlation with clinico-pathological parameters, expression of Hsp27 was linked to alpha-fetoprotein level (P = 0.007) whereas up-regulation of GRP78 was associated with tumor venous infiltration (P = 0.035). No significant association of Hsp70 with any pathologic features was observed. The present HCC proteome analysis revealed that in response to the stressful cancerous microenvironment, tumor cells strived to increase the expression of chaperone proteins for cyto-protective function and to enhance tumor growth and metastasis.     

Optimization of a phenol extraction‐based protein preparation method amenable to downstream 2DE and MALDI‐MS based analysis of bacterial proteomes

2DE is one of the most efficient and widely used methods for resolving complex protein mixtures. For efficient analysis of complex samples, high‐resolution separation of proteins on 2D gel is essential, and for that purpose good sample preparation is crucial. In this study, we have improvized a method for preparing bacterial total cellular proteome, from a strategy applied earlier to recalcitrant plant tissues, which gave high‐quality resolution on 2DE. The method involving phenol extraction followed by methanol/ammonium acetate precipitation was first optimized for the chemolithotrophic proteobacteria Tetrathiobacter kashmirensis WT001 and Pseudaminobacter salicylatoxidans KCT001 that did not yield quality protein preps in conventional trichloroacetic acid/acetone precipitation method. Subsequently, to validate its general applicability, the method was evaluated against the trichloroacetic acid/acetone precipitation method for two other model bacteria, i.e. Escherichia coli DH5α and Mycobacterium smegmatis mc²6. Identification of at least four proteins each from the outer membrane, periplasm, and cytoplasm of T. kashmirensis by MALDI‐MS not only proved the efficiency of the method in extracting proteins from the different cellular compartments but also the amenability of the obtained protein spots toward MALDI‐MS based identification.     

Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host

This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection.     

Human urine proteome analysis by three separation approaches

The urinary proteome is known to be a valuable field of study related to organ functions. There have been several extensive urine proteome studies. However, the overlapping rate among different studies is relatively low. Whether the low overlapping rate was caused by different sample sources, preparation, separation and identification methods is unknown. Moreover, low molecular mass (<10 kDa) proteins have not been studied extensively. In this report, male and female pooled urine samples were collected from healthy volunteers. The urinary proteins were acetone precipitated, separated and identified by three approaches, 1-DE plus 1-D LC/MS/MS, direct 1-D LC/MS/MS and 2-D LC/MS/MS. 1-D tricine gels were used to separate low molecular mass proteins. The tandem mass spectra of positive identifications were quality controlled both by manual validation and using advanced mass spectrum scanner software. A total of 226 urinary proteins were identified; 171 proteins were identified by proteomics approach for the first time, including 4 male-specific proteins. Twelve low molecular mass proteins were identified. Most urinary proteins had a molecular mass between 30 and 60 kDa and a pI between 4 and 10. The apparent molecular masses of many proteins were different from theoretical ones, which indicated their post-translational modification and degradation. The effects of sample preparation, separation and identification methods on the overlapping rate of different experiments are discussed.     

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.     

Expanding the mouse embryonic stem cell proteome: Combining three proteomic approaches

The current study used three different proteomic strategies, which differed by their extent of intact protein separation, to examine the proteome of a pluripotent mouse embryonic stem cell line, R1. Proteins from whole‐cell lysates were subjected either to 2‐D‐LC, or 1‐DE, or were unfractionated prior to enzymatic digestion and subsequent analysis by MS. The results yielded 1895 identified non‐redundant proteins and, for 128 of these, the specific isoform could be determined based on detection of an isoform‐specific peptide. When compared with two previously published proteomic studies that used the same cell line, the current study reveals 612 new proteins.     

Benefits of heat treatment to the protease packed neutrophil for proteome analysis: halting protein degradation

Neutrophils, cells of the innate immune system, contain an array of proteases and reactive oxygen species-generating enzymes that assist in controlling the invasion of bacteria and pathogens. The high content of intracellular proteolytic enzymes makes them difficult cells to work with as they can degrade proteins of potential interest. Here, we describe the benefits of heat treatment of neutrophils in reducing protein degradation for subsequent proteome analysis. Neutrophils isolated from four healthy volunteers were each divided into three aliquots and subjected to different preparation methods for 2-DE: (i) Heat treatment, (ii) resuspension in NP40 lysis buffer and (iii) resuspension in standard 2-DE lysis buffer. Representative spots found to be statistically significant between groups (p<0.01) were excised and identified by LC-MS/MS, three of which were validated by immunoblotting. Heat-treated samples contained proteins in the high-molecular-weight range that were absent from NP40-treated samples. Moreover, NP40-treated samples showed an increase in spot number and volume at lower molecular weights suggestive of protein degradation. Incorporating heat treatment into sample preparation resulted in the identification of proteins that may not have previously been detected due to sample degradation, thus leading to a more comprehensive 2-DE map of the human neutrophil proteome.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

A Proteomic approach for protein-profiling the oncogenic ras induced transformation (H-, K-, and N-Ras) in NIH/3T3 mouse embryonic fibroblasts

Point mutations in three kinds of Ras protein (H-, K-, and N-Ras) that specifically occur in codons 12, 13, and 61 facilitate virtually all of the malignant phenotype of the cancer cells, including cellular proliferation, transformation, invasion, and metastasis. In order to elucidate an understanding into the oncogenic ras networks by H-, K-, and N-Ras/G12V, we have established various oncogenic ras expressing NIH/3T3 mouse embryonic fibroblast clones using the tetracycline-induction system, which are expressing Ras/G12V proteins under the tight control of expression by an antibiotics, doxycycline. Here we provide a catalog of proteome profiles in total cell lysates derived from three oncogenic ras expressing NIH/3T3 cells and a good in vitro model system for dissecting the protein networks due to these oncogenic Ras proteins. In this biological context, we compared total proteome changes by the combined methods of 2-DE, quantitative image analysis, and MALDI-TOF MS analysis using the unique Tet-on inducible expression system. There were a large number of common targets for oncogenic ras, which were identified in all three cell lines and consisted of 204 proteins (61 in the pH range of 4-7, 63 in 4.5-5.5, and 80 in 5.5-6.7). Differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. Taken together, we implemented a 2-DE-based proteomics approach to the systematical analysis of the dysregulations in the cellular proteome of NIH/3T3 cells transformed by three kinds of oncogenic ras. Our results obtained and presented here show that the comparative analysis of proteome from oncogenic ras expressing cells has yielded interpretable data to elucidate the differential protein expression directly and/or indirectly, and contributed to evaluate the possibilities for physiological, and therapeutic targets. Further studies are in progress to elucidate the implications of these findings in the regulation of Ras induced transformation.     

Proteomic analysis of chlorosome-depleted membranes of the green sulfur bacterium Chlorobium tepidum

Green sulfur bacteria are obligate anaerobic phototrophs, which in addition to outer and plasma membranes contain chlorosomes. The analysis of the membrane proteome of Chlorobium tepidum from chlorosome-depleted membranes is described in this study. The membranes were purified by sucrose density centrifugation and characterized by 1-DE and 2-DE coupled with MS, absorption spectroscopy, and electron microscopy. 1-DE and 2-DE were employed to analyze the membrane proteins and to characterize the capabilities of the methods. Solubilization of the membrane proteins prior to 2-DE was improved by using a series of zwitterionic detergents. Based on the resolved spots after 2-DE, the combination of amidosulfobetaine 14 with Triton X-100 is more efficient than the combination of CHAPS, N-decyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, and Triton X-100. From the application of 1-DE and 2-DE, 167 and 202 unique proteins were identified, respectively, using PMF by MALDI-TOF MS. Both methods resulted in the detection of 291 different proteins of which only 88 were predicted membrane proteins, indicating the limitation of membrane protein detection after separation with electrophoresis methods. In addition, 53 of these proteins were identified as outer membrane proteins.     

Protein profile of exosomes from trabecular meshwork cells

To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to a two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure.     

Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques

Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a unique set of integral and peripherally associated proteins. To obtain a complete protein inventory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral membrane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate that the combination of all three methods provides a comprehensive survey of the proteinaceous inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle proteins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide triphosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our results demonstrate that the parallel application of three different gel-based approaches in combination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle proteome that is considerably more complex than previously anticipated.     

A comprehensive proteome map of bovine cerebrospinal fluid

Cerebrospinal fluid (CSF) is considered as the most promising body fluid target for the discovery of biomarkers for early diagnosis of neurodegenerative diseases such as Creutzfeldt–Jakob disease in humans and bovine spongiform encephalopathy in cattle. For the recognition of disease‐associated changes in bovine CSF protein patterns, a detailed knowledge of this proteome is a prerequisite. The absence of a high‐resolution CSF proteome map prompted us to determine all bovine CSF protein spots that can be visualised on 2‐D protein gels. Using state‐of‐the‐art 2‐DE technology for proteome mapping of bovine ante mortem CSF combined with sensitive fluorescent protein staining and MALDI‐TOF/TOF MS for protein identification, a highly detailed 2‐DE map of the bovine CSF proteome was established. Besides the proteins mapped by earlier studies, this map contains 66 different proteins, including 58 which were not annotated in bovine 2‐DE CSF maps before.