An investigation into the human serum "interactome"

The protein content of human serum is composed of a millieu of proteins from almost every type of cell and tissue within the body. The serum proteome has been shown to contain information that directly reflects pathophysiological states and represents an invaluable source of diagnostic information for a variety of different diseases. Unfortunately, the dynamic range of protein abundance, ranging from > mg/mL level to < pg/mL level, renders complete characterization of this proteome nearly impossible with current analytical methods. To study low-abundance proteins, which have potential value for clinical diagnosis, the high-abundant species, such as immunoglobulins and albumin, are generally eliminated as the first step in many analytical protocols. This step, however, is hypothesized to concomitantly remove proteins/peptides associated with the high-abundant proteins targeted for depletion. In this study, immunoprecipitation was combined with microcapillary reversed-phase liquid chromatography (microRPLC) coupled on-line with tandem mass spectrometry (MS/MS) to investigate the low-molecular-weight proteins/peptides that associate with the most abundant species in serum. By this targeted isolation of select highly abundant serum proteins, the associated proteins/peptides can be enriched and effectively identified by microRPLC-MS/MS. Among the 210 proteins identified, 73% and 67% were not found in previous studies of the low-molecular-weight or whole-serum proteome, respectively.     

Novel application of Ag nanoclusters in fluorescent imaging of human serum proteins after native polyacrylamide gel electrophoresis (PAGE)

We have developed a novel application for DNA oligonucleotide-stabilized Ag nanoclusters in fluorescent imaging of human serum proteins after native polyacrylamide gel electrophoresis (PAGE). Oligonucleotide-stabilized Ag nanoclusters were used as fluorescent probes for direct detection of proteins after native PAGE. Some relatively low-abundance proteins, such as α-1-antichymotrypsin (ACT) and α-2-glycoprotein 1, zinc (ZAG) were easily detected by oligonucleotide-stabilized Ag nanocluster-based fluorescent imaging and identified by MS and MS/MS techniques, without the need of expensive antibodies or tedious immunoassay procedures. The pH condition for the oligonucleotide-stabilized Ag nanocluster solution was optimized and the possible mechanism of interaction between proteins and DNA oligonucleotide-stabilized Ag nanoclusters was analyzed. As a novel fluorescent detection method it is simple, fast, nontoxic and sensitive, and it shows great analytical potential in proteome research and in biochemistry.     

Comprehensive analysis of low-abundance proteins in human urinary exosomes using peptide ligand library technology, peptide OFFGEL fractionation and nanoHPLC-chip-MS/MS

Human urinary exosomes are 30-100 nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary track and may serve as a suitable noninvasive starting material for biomarker discovery relevant to a variety of renal disease. To comprehensively explore the low-abundance proteome, combinatorial peptide ligand libraries, combined with peptide OFFGEL electrophoresis were employed for the enrichment and separation of relatively low-abundant proteins in urinary exosomes. After analysis by nanoHPLC-chip-MS/MS, 512 proteins were identified, including a large number of proteins with extreme molecular weight or extreme pI value, which could not be well mapped by using traditional 2-D-gel-based separation methods. This in-depth analysis of low-abundant proteins in urinary exosomes led to an increased understanding of molecular composition of these little vesicles and may be helpful for the discovery of novel biomarker. Our work also provides an effective strategy of concentration and identification of low-abundance proteome from complex bio-samples.     

液相等电聚焦结合双向凝胶电泳分离碱性蛋白

在蛋白组学研究中, 经典的双向凝胶电泳法(2-DE)对碱性蛋白及低丰度蛋白的分离存在技术障碍, 但预分离技术的应用可弥补其缺陷. 液相等电聚焦可有效地分离富集复杂蛋白样品. 碱性胶条用于2-DE可极大地提高蛋白上样量和凝胶分辨率. 将上述两种技术相结合用于碱性蛋白质和低丰度蛋白质的分离鉴定, 可使碱端区域双向凝胶图谱质量显著提高, 蛋白点更清晰且点数增多, 质谱鉴定确信度提高, 碱性蛋白和低丰度蛋白质谱鉴定成功率提高, 对于蛋白组学研究具有一定的意义.     

Comprehensive proteome analysis of mouse liver by ampholyte-free liquid-phase isoelectric focusing

In this study, ampholyte-free liquid-phase IEF (LIEF) was combined with narrow pH range 2-DE and SDS-PAGE RP-HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2-DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2-DE gels following LIEF prefractionation into a single virtual 2-DE gel. Furthermore, the pH 3-5 LIEF fraction and the unfractionated sample were separated by pH 3-6 2-DE and identified by MALDI-TOF/TOF MS, respectively. In parallel, the pH 3-5 LIEF fraction was also analyzed by SDS-PAGE RP-HPLC MS/MS. LIEF-2-DE and LIEF-HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low-abundance proteins and proteins with extreme physicochemical characteristics or post-translational modifications compared to conventional 2-DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large-scale datasets. It was observed that the combination of LIEF-2-DE and LIEF-HPLC was effective in promoting MS-based liver proteome profiling and could be applied on similar complex tissue samples.     

Proteomics: the move to mixtures.

Proteomics can be defined as the systematic analysis of proteins for their identity, quantity and function. In contrast to a cell's static genome, the proteome is both complex and dynamic. Proteome analysis is most commonly accomplished by the combination of two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). However, this technique is under scrutiny because of a failure to detect low-abundance proteins from the analysis of whole cell lysates. Alternative approaches integrate a diversity of separation technologies and make use of the tremendous peptide separation and sequencing power provided by MS/MS. When liquid chromatography is combined with tandem mass spectrometry (LC/MS/MS) and applied to the direct analysis of mixtures, many of the limitations of 2DE for proteome analysis can be overcome. This tutorial addresses current approaches to identify and characterize large numbers of proteins and measure dynamic changes in protein expression directly from complex protein mixtures (total cell lysates).     

Quantitative analysis of low-abundance serological proteins with peptide affinity-based enrichment and pseudo-multiple reaction monitoring by hybrid quadrupole time-of-flight mass spectrometry

Multiple reaction monitoring (MRM) is commonly used for the quantitative analysis of proteins during mass pectrometry (MS), and has excellent specificity and sensitivity for an analyte in a complex sample. In this study, a pseudo-MRM method for the quantitative analysis of low-abundance serological proteins was developed using hybrid quadrupole time-of-flight (hybrid Q-TOF) MS and peptide affinity-based enrichment. First, a pseudo-MRM-based analysis using hybrid Q-TOF MS was performed for synthetic peptides selected as targets and spiked into tryptic digests of human serum. By integrating multiple transition signals corresponding to fragment ions in the full scan MS/MS spectrum of a precursor ion of the target peptide, a pseudo-MRM MS analysis of the target peptide showed an increased signal-to-noise (S/N) ratio and sensitivity, as well as an improved reproducibility. The pseudo-MRM method was then used for the quantitative analysis of the tryptic peptides of two low-abundance serological proteins, tissue inhibitor of metalloproteinase 1 (TIMP1) and tissue-type protein tyrosine phosphatase kappa (PTPκ), which were prepared with peptide affinity-based enrichment from human serum. Finally, this method was used to detect femtomolar amounts of target peptides derived from TIMP1 and PTPκ, with good coefficients of variation (CV 2.7% and 9.8%, respectively), using a few microliters of human serum from colorectal cancer patients. The results suggest that pseudo-MRM using hybrid Q-TOF MS, combined with peptide affinity-based enrichment, could become a promising alternative for the quantitative analysis of low-abundance target proteins of interest in complex serum samples that avoids protein depletion.     

Isolation, identification and characterisation of starch-interacting proteins by 2-D affinity electrophoresis

A 2-D affinity electrophoretic technique (2-DAE) has been used to isolate proteins that interact with various starch components from total barley endosperm extracts. In the first dimension, proteins are separated by native PAGE. The second-dimensional gel contains polysaccharides such as amylopectin and glycogen. The migration of starch-interacting proteins in this dimension is determined by their affinity towards a particular polysaccharide and these proteins are therefore spatially separated from the bulk of proteins in the crude extract. Four distinct proteins demonstrate significant affinity for amylopectin and have been identified as starch branching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa), SBEIIb and starch phosphorylase using polyclonal antibodies and zymogram activity analysis. In the case of starch phosphorylase, a protein spot was excised from a 2-DAE polyacrylamide gel and analysed using Q-TOF MS/MS, resulting in the alignment of three internal peptide sequences with the known sequence of the wheat plastidic starch phosphorylase isoform. This assignment was confirmed by the determination of the enzyme's function using zymogram analysis. Dissociation constants (Kd) were calculated for the three enzymes at 4 degrees C and values of 0.20, 0.21 and 1.3 g/L were determined for SBEI, SBEIIa and starch phosphorylase, respectively. Starch synthase I could also be resolved from the other proteins in the presence of glycogen and its identity was confirmed using a polyclonal antibody and by activity analysis. The 2-DAE method described here is simple, though powerful, enabling protein separation from crude extracts on the basis of function.     

Comparison of surfactant-assisted shotgun methods using acid-labile surfactants and sodium dodecyl sulfate for membrane proteome analysis

Three surfactant-assisted shotgun methods using acid labile surfactants, sodium-3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)-methoxyl]-1-propanesulfonate (RapiGest) and 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), and sodium dodecyl sulfate (SDS) were investigated for their applicability to membrane proteome analysis. It is shown that RapiGest is a preferred reagent for handling membrane proteomes of Escherichia coli and MCF7 cells for liquid chromatography tandem mass spectrometry (LC MS/MS) analysis of tryptic digests. The RapiGest method allowed identification of more peptides and proteins than the SDS and PPS methods and there was no apparent bias for the type of peptides and proteins identified by the RapiGest and SDS methods, while a slightly higher proportion of hydrophilic peptides and proteins were identified by the PPS method. The performance of the SDS and PPS methods is similar in terms of the numbers of peptides and proteins identified. Since the SDS method required the removal of SDS using a technique such as strong-cation exchange (SCX), we further investigated the effect of SCX on sample loss through analyzing the digest of an enriched E. coli membrane fraction as well as a standard protein, bovine serum albumin (BSA). The results showed that extensive sample loss (as much as 62%) was encountered during the SCX cleaning step. We then applied the RapiGest method in combination with two-dimensional LC MS/MS to characterize the E. coli membrane proteome. In total, 1626 unique proteins (5799 unique peptides) were identified with a peptide false discovery rate of 2.4%. About 60% of the identified proteins with known cellular locations were found to be membrane proteins. Among them, about 75% were integral membrane proteins. This work represents one of the most comprehensive profiles of E. coli membrane proteome generated by a proteomic technique.     

Evaluation of sample extraction methods for proteomics analysis of green algae Chlorella vulgaris

Many protein extraction methods have been developed for plant proteome analysis but information is limited on the optimal protein extraction method from algae species. This study evaluated four protein extraction methods, i.e. direct lysis buffer method, TCA‐acetone method, phenol method, and phenol/TCA‐acetone method, using green algae Chlorella vulgaris for proteome analysis. The data presented showed that phenol/TCA‐acetone method was superior to the other three tested methods with regards to shotgun proteomics. Proteins identified using shotgun proteomics were validated using sequential window acquisition of all theoretical fragment‐ion spectra (SWATH) technique. Additionally, SWATH provides protein quantitation information from different methods and protein abundance using different protein extraction methods was evaluated. These results highlight the importance of green algae protein extraction method for subsequent MS analysis and identification.     

A 2D reversed-phase × ion-pair reversed-phase HPLC-MALDI TOF/TOF-MS approach for shotgun proteome analysis

The separation of complex peptide mixtures in shotgun proteome analysis using a 2D separation scheme encompassing reversed-phase × ion-pair reversed-phase (IP-RP) liquid chromatography coupled online to electrospray ion trap mass spectrometry (MS) has been shown earlier to be superior in terms of separation efficiency and technical robustness compared to the classically used separation scheme encompassing strong cation exchange × IP-RP-chromatography in shotgun proteome analysis. In the present study, this novel separation scheme was coupled offline to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF-MS for the analysis of the same sample, a tryptic digest of the cytosolic proteome of the bacterium Corynebacterium glutamicum. Compared to the earlier study, the MALDI-based platform led to a significantly increased number of peptides (7,416 vs. 2,709) and proteins (1,208 vs. 468, without single peptide-based identifications), respectively. This represents the majority of all predicted cytosolic proteins in C. glutamicum. The high proteome coverage, as well as the large number of low-abundant proteins identified with this improved analytical platform, pave the way for new biological studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

2-DE proteomic analysis of the model cyanobacterium Anabaena variabilis

Cyanobacteria are photosynthetic bacteria capable of producing hydrogen and secondary metabolites with potential pharmaceutical applications. A limited number of cyanobacterial 2-DE proteomic studies have been published, most of which are based on Synechocystis sp. PCC 6803. Here, we report the use of 2-DE, ESI-MS/MS and protein bioinformatics tools to characterize the proteome of Anabaena variabilis ATCC 29413, a heterocystous nitrogen-fixing cyanobacterium that is a model organism for the study of nitrogen fixation. Using a 2-DE workflow that included the use of a detergent-based extraction buffer and 3-10 nonlinear IPG strips resulted in the identification of 254 unique proteins, with significantly better coverage of basic and low-abundance proteins that has been reported in 2-DE analyses of Synechocystis sp. A set of protein bioinformatics tools was employed to provide estimates of protein localization, hydrophobicity, abundance and other properties. The characteristics of the A. variabilis proteins identified in this study were compared against the theoretical proteome for this organism, and more generally within the cyanobacteria, to identify opportunities for further development of 2-DE-based cyanobacterial proteomics.     

Continuous sample deposition from reversed-phase liquid chromatography to tracks on a matrix-assisted laser desorption/ionization precoated target for the analysis of protein digests

Peptide mass fingerprinting by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) is one of the standard high-throughput methods for protein identification today. Traditionally this method has been based on spotting peptide mixtures onto MALDI targets. While this method works well for more abundant proteins, low-abundance proteins mixed with high-abundance proteins tend to go undetected due to ion suppression effects, instrumental dynamic range limitations and chemical noise interference. We present an alternative approach where liquid chromatography (LC) effluent is continuously collected as linear tracks on a MALDI target. In this manner the chromatographic separation is spatially preserved on the target, which enables generation of off-line LC-MS and LC-MS/MS data by MALDI. LC-MALDI sample collection provides improved sensitivity and dynamic range, spatial resolution of peptides along the sample track, and permits peptide mass mapping of low-abundance proteins in mixtures containing high-abundance proteins. In this work, standard and ribosomal protein digests are resolved and captured using LC-MALDI sample collection and analyzed by MALDI-TOF-MS.     

Combination of FASP and fully automated 2D‐LC‐MS/MS allows in‐depth proteomic characterization of mouse zymogen granules

Zymogen granule (ZG) constituents play important roles in pancreatic injury and disease. In previous studies, proteomic analyses with rat zymogen granules were separated by two‐dimensional gel electrophoresis or one‐dimensional SDS–PAGE, followed by in‐gel tryptic digestion. In order to overcome the disadvantage of in‐gel digestion and to carry out further in‐depth proteomic analysis of the zymogen granules, in this study, by combining a filter‐aided sample preparation method and fully automated 2D‐LC‐MS/MS technique, 800 ZG proteins were identified with at least two unique peptides for each protein, 75% of which have not been previously reported. The identified proteins revealed broad diversity in protein identity and function. This is the largest dataset of ZG proteome, and also the first dataset of the mouse ZG proteome, which may help elucidate on the molecular architecture of ZGs and their functions. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of low-abundance proteins of bovine colostral and mature milk using two-dimensional electrophoresis followed by microsequencing and mass spectrometry

We identified several low-abundance proteins of bovine colostrum and mature milk using the immunoabsorption technique and two-dimensional electrophoresis (2-DE) followed by microsequencing and mass spectrometry. Two major milk proteins, beta-casein and immunoglobulin G (IgG), were effectively removed from the milk using immunoabsorbents. Milk samples before and after immunoabsorption were separated by 2-DE. Protein identification of the spots on 2-DE was performed by either gel comparison, microsequencing, matrix-assisted laser desorption/ionization-time of flight mass-spectrometry (MALDI-TOF-MS), peptide mass fingerprinting or peptide sequencing using tandem MS by hybrid quadrupole/orthogonal acceleration time of flight-MS (Q-TOF). Significant differences in protein patterns were observed between the low-abundance proteins of colostrum and mature milk. In addition, several low-abundance proteins including fibrinogen beta-chain, chitinase 3-like 1, alpha-antitrypsin, complement C3 alpha-chain, gelsolin and apolipoprotein H were observed only in colostrum. However, the level of beta-casein fragments increased significantly during this lactation period. alpha-Lactalbumin and beta-lactoglobulin as well as some low-abundance proteins including bovine serum albumin, serotransferrin and lactoferrin were identified in both colostral and mature milk. Low-abundance proteins in bovine colostrum may have special physiologic relevance to the health and development of calves early in lactation.     

Sugarcane proteomics: Establishment of a protein extraction method for 2‐DE in stalk tissues and initiation of sugarcane proteome reference map

Sugarcane is an important commercial crop cultivated for its stalks and sugar is a prized commodity essential in human nutrition. Proteomics of sugarcane is in its infancy, especially when dealing with the stalk tissues, where there is no study to date. A systematic proteome analysis of stalk tissue yet remains to be investigated in sugarcane, wherein the stalk tissue is well known for its rigidity, fibrous nature, and the presence of oxidative enzymes, phenolic compounds and extreme levels of carbohydrates, thus making the protein extraction complicated. Here, we evaluated five different protein extraction methods in sugarcane stalk tissues. These methods are as follows: direct extraction using lysis buffer (LB), TCA/acetone precipitation followed by solubilization in LB, LB containing thiourea (LBT), and LBT containing tris, and phenol extraction. Both quantitative and qualitative protein analyses were performed for each method. 2‐DE analysis of extracted total proteins revealed distinct differences in protein patterns among the methods, which might be due to their physicochemical limitations. Based on the 2‐D gel protein profiles, TCA/acetone precipitation‐LBT and phenol extraction methods showed good results. The phenol method showed a shift in pI values of proteins on 2‐D gel, which was mostly overcome by the use of 2‐D cleanup kit after protein extraction. Among all the methods tested, 2‐D cleanup‐phenol method was found to be the most suitable for producing high number of good‐quality spots and reproducibility. In total, 30 and 12 protein spots commonly present in LB, LBT and phenol methods, and LBT method were selected and subjected to eLD‐IT‐TOF‐MS/MS and nESI‐LC‐MS/MS analyses, respectively, and a reference map has been established for sugarcane stalk tissue proteome. A total of 36 nonredundant proteins were identified. This is a very first basic study on sugarcane stalk proteome analysis and will promote the unexplored areas of sugarcane proteome research.     

Proteome profile of zebrafish caudal fin based on one-dimensional gel electrophoresis LCMS/MS and two-dimensional gel electrophoresis MALDI MS/MS analysis

Zebrafish (Danio rerio) is the widely used vertebrate model animal for understanding the complexity of development and disease process. Zebrafish has been also extensively used in understanding the mechanism of regeneration for its extensive capability of regenerating fins and other tissues. We have analyzed the proteome profile of zebrafish caudal fin in its native state based on one-dimensional gel electrophoresis LCMS/MS and two-dimensional gel electrophoresis MS/MS analyses. A total of 417 proteins were identified as zebrafish fin tissue specific, which includes 397 proteins identified based on one-dimensional gel electrophoresis LCMS/MS analysis and 101 proteins identified based on two-dimensional gel electrophoresis MALDI MS/MS. The proteins mapped to the zebrafish fin tissue were shown to be involved in various biological activities related to development, apoptosis, signaling and metabolic process. Focal adhesion, regulation of actin cytoskeleton, cancer-related pathways, mitogen-activated protein kinase signaling, antigen processing and presentation, and proteasome are some of the important pathways associated with the identified proteome data set of the zebrafish fin.     

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

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