mTRAQ-based quantitative analysis combined with peptide fractionation based on cysteinyl peptide enrichment

In the present study, the fractionation scheme for cysteinyl peptide enrichment (CPE) was combined with the mass differential tags for relative and absolute quantification (mTRAQ) method to reduce sample complexity and increase proteome coverage. Cysteine residues of the proteins were first alkylated using iodoacetyl PEG₂–biotin instead of other conventional alkylating agents such as iodoacetamide. After trypsin digestion, amine groups were labeled with mTRAQ, and these labeled peptides were fractionated according to the presence or absence of cysteine residues using avidin–biotin affinity chromatography. With these approaches, we were able to divide the peptides into the two fractions with more than 90% fractionation efficiency for standard protein and MCF7 cell lysate. When the fractionation strategy was applied to colorectal cancer tissue samples, we were able to obtain quantitative information that was consistent with the previous study based on mTRAQ quantification, implying that the cysteine-based fractionation method does not affect mTRAQ quantification. We expect that the mTRAQ-based quantitative analysis combined with peptide fractionation through the CPE strategy would allow for deep-down analysis of proteome samples and ultimately for increasing proteome coverage with simultaneous quantification for biomarker discovery.     

Increased proteome coverage by combining PAGE and peptide isoelectric focusing: Comparative study of gel‐based separation approaches

The in‐depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC‐MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in‐gel IEF, prior to RP‐HPLC‐MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.     

Proteomics analysis of cells in whole saliva from oral cancer patients via value-added three-dimensional peptide fractionation and tandem mass spectrometry

Whole human saliva possesses tremendous potential in clinical diagnostics, particularly for conditions within the oral cavity such as oral cancer. Although many have studied the soluble fraction of whole saliva, few have taken advantage of the diagnostic potential of the cells present in saliva, and none have taken advantage of proteomics capabilities for their study. We report on a novel proteomics method with which we characterized for the first time cells contained in whole saliva from patients diagnosed with oral squamous cell carcinoma. Our method uses three dimensions of peptide fractionation, combining the following steps: preparative IEF using free flow electrophoresis, strong cation exchange step gradient chromatography, and microcapillary reverse-phase liquid chromatography. We determined that the whole saliva samples contained enough cells, mostly exfoliated epithelial cells, providing adequate amounts of total protein for proteomics analysis. From a mixture of four oral cancer patient samples, the analysis resulted in a catalogue of over 1000 human proteins, each identified from at least two peptides, including numerous proteins with a role in oral squamous cell carcinoma signaling and tumorigenesis pathways. Additionally proteins from over 30 different bacteria were identified, some of which putatively contribute to cancer development. The combination of preparative IEF followed by strong cation exchange chromatography effectively fractionated the complex peptide mixtures despite the closely related physiochemical peptide properties of these separations (pI and solution phase charge, respectively). Furthermore compared with our two-step method combining preparative IEF and reverse-phase liquid chromatography, our three-step method identified significantly more cellular proteins while retaining higher confidence protein identification enabled by peptide pI information gained through IEF. Thus, for detecting salivary markers of oral cancer and possibly other conditions of the oral cavity, the results confirm both the potential of analyzing the cells in whole saliva and doing so with our proteomics method.     

Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis

The sample fractionation steps conducted prior to mass detection are critically important for the comprehensive analysis of complex protein mixtures. This paper illustrates the effectiveness of OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator for the fractionation of peptides. An Escherichia coli tryptic digest was separated in 24 fractions, and peptides were identified by reversed-phase liquid chromatography on a microfluidic device with mass spectrometric detection. About 90% of the identified individual peptides were found in only one or two fractions. The distribution of the calculated isoelectric points for the peptides identified in each fraction was especially narrow in the acidic pH range. Standard deviations approached the size of the pH segment covered by the respective fraction. The experimental peptide isoelectric point measured by OFFGEL electrophoresis was used as an additional filter for validation of peptide identifications.     

Improved proteome coverage by using iTRAQ labelling and peptide OFFGEL fractionation

Background  

The development of mass spectrometric techniques and fractionation methods now allows the investigation of very complex protein mixtures ranging from subcellular structures to tissues. Nevertheless, this work is particularly difficult due to the wide dynamic range of protein concentration in eukaryotic tissues. In this paper, we present a shotgun method whereby the peptides are fractionated using OFFGEL electrophoresis after iTRAQ labelling.     

Studies on peptide acetylation for stable-isotope labeling after 1-D PAGE separation in quantitative proteomics

Acetylation is a single labeling process to label peptides in control and experimental samples universally, and is independent of amino acid composition or post-translational modification. Here, we propose a new strategy especially useful to quantify either hydrophobic or extremely acidic and basic proteins involved in acetylation of tryptic peptides after sodium dodecyl sulfate polyarcylamide gel electrophoresis (SDS-PAGE) separation. We studied some essential parameters of acetylation labeling reactions in either in-solution tryptic peptides or in-gel digested extracts systematically. We have found that the acetylation efficiency varies markedly on account of different reactive systems, and demonstrated that stable isotope labeling can be steadily obtained with in-gel digested peptides under optimized conditions. We use this protocol to quantify some proteins of two kinds of hepatocellular carcinoma cell line, non-metastatic hepatocellular carcinoma cells, Hep3B, and metastatic hepatocellular carcinoma cells, MHCC97-H. The experimental results provide positive evidence for the potential application of an acetylation labeling strategy in quantitative proteomics, and an efficient way for global proteome quantification.     

Proteomics for biodeterioration of wood (Pinus taeda L.): Challenging analysis by 2-D PAGE and MALDI-TOF/TOF/MS

Proteins expressed by the brown-rot fungus Gloeophyllum trabeum were characterized from inoculated southern yellow pine sapwood undergoing decay, from pure cultures of the fungus and from uninoculated pinewood. Analysis was carried out by two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF/TOF/MS. No proteins were detected from the clean uncontaminated wood. The inoculated wood undergoing active brown-rot decay produced 76 proteins, including the Fenton-chemistry related enzymes, alcohol oxidase, lipoxygenase, and catalase. One hundred and eleven proteins were detected from the pure culture and most were common metabolic proteins. A majority of proteins in both samples were identified as hypothetical proteins. A surprising result is that there was very little overlap between proteins found in both sets of samples, indicating a very different mechanism in action when the fungus is growing on a cellulose-based nutrient source (wood) versus glucose media. This study also highlights a current limitation of this approach, which is the limited protein and genomic sequence information annotated on the public databases. Of the 187 proteins characterized, only 36 were identified with confidence. To our knowledge, this is the first reported proteomic analysis of pinewood decayed by a brown-rot fungus and provides the initial characterization of proteins involved in this type of wood biodeterioration. Although significant limitations still exist in identifying the proteins, this limitation will diminish as functional proteins are identified and added to the databases.     

5个生姜品种的蛋白电泳指纹图谱研究

目的：研究5个生姜品种的鉴定。方法：利用蛋白质聚丙烯酰胺凝胶电泳（PAGE）指纹图谱分析、鉴别5个生姜品种。结果：5个样品的蛋白质PAGE指纹图谱具有一定差异。结论：PAGE指纹图谱可用于生姜品种鉴定与质量评价。     

An alternative strategy to determine the mitochondrial proteome using sucrose gradient fractionation and 1D PAGE on highly purified human heart mitochondria

An alternative strategy for mitochondrial proteomics is described that is complementary to previous investigations using 2D PAGE techniques. The strategy involves (a) obtaining highly purified preparations of human heart mitochondria using metrizamide gradients to remove cytosolic and other subcellular contaminant proteins; (b) separation of mitochondrial protein complexes using sucrose density gradients after solubilization with n-dodecyl-beta-D-maltoside; (c) 1D electrophoresis of the sucrose gradient fractions; (d) high-throughput proteomics using robotic gel band excision, in-gel digestion, MALDI target spotting and automated spectral acquisition; and (e) protein identification from mixtures of tryptic peptides by high-precision peptide mass fingerprinting. Using this approach, we rapidly identified 82 bona fide or potential mitochondrial proteins, 40 of which have not been previously reported using 2D PAGE techniques. These proteins include small complex I and complex IV subunits, as well as very basic and hydrophobic transmembrane proteins such as the adenine nucleotide translocase that are not recovered in 2D gels. The technique described here should also be useful for the identification of new protein-protein associations as exemplified by the validation of a recently discovered complex that involves proteins belonging to the prohibitin family.     

基于质谱的定量蛋白质组学策略和方法研究进展

定量蛋白质组学已经成为组学领域研究的热点之一.相关实验技术和计算方法的不断创新极大地促进了定量蛋白质组学的飞速发展.常用的定量蛋白质组学策略按照是否需要稳定同位素标记可以分为无标定量和有标定量两大类.每类策略又产生了众多定量方法和工具,它们一方面推动了定量蛋白质组学的深入发展;另一方面,也在实验策略与技术的发展过程中不断更新.因此对这些定量实验策略和方法进行系统总结和归纳将有助于定量蛋白质组学的研究.本文主要从方法学角度全面归纳了目前定量蛋白质组学研究的相关策略和算法,详述了无标定量和有标定量的具体算法流程并比较了各自特点,还对以研究蛋白质绝对丰度为目标的绝对定量算法进行了总结,列举了常用的定量软件和工具,最后概述了定量结果的质量控制方法,对定量蛋白质组学方法发展的前景进行了展望.     

Proteomics shows Hsp70 does not bind peptide sequences indiscriminately in vivo

Heat shock protein 70 (Hsp70) binds peptide and has several functions that include protein folding, protein trafficking, and involvement with immune function. However, endogenous Hsp70-binding peptides had not previously been identified. Therefore, we eluted and identified several hundred endogenously bound peptides from Hsp70 using liquid chromatography ion trap mass spectrophotometry (LC-ITMS). Our work shows that the peptides are capable of binding Hsp70 as previously described. They are generally 8-26 amino acids in length and correspond to specific regions of many proteins. Through computationally assisted analysis of peptides eluted from Hsp70 we determined variable amino acid sequences, including a 5 amino acid core sequence that Hsp70 favorably binds. We also developed a computer algorithm that predicts Hsp70 binding within proteins. This work helps to define what peptides are bound by Hsp70 in vivo and suggests that Hsp70 facilitates peptide selection by aiding a funneling mechanism that is flexible but allows only a limited number of peptides to be processed.     

Continuous flow magnetic cell fractionation based on antigen expression level

Cell separation is important in medical and biological research and plays an increasingly important role in clinical therapy and diagnostics, such as rare cancer cell detection in blood. The immunomagnetic labeling of cells with antibodies conjugated to magnetic nanospheres gives rise to a proportional relationship between the number of magnetic nanospheres attached to the cell and the cell surface marker number. This enables the potential fractionation of cell populations by magnetophoretic mobility (MM). We exploit this feature with our apparatus, the Dipole Magnet Flow Fractionator (DMFF), which consists of an isodynamic magnetic field, an orthogonally-oriented thin ribbon of cell suspension in continuous sheath flow, and ten outlet flows. From a sample containing a 1:1 mixture of immunomagnetically labeled (label+) and unlabeled (label-) cells, we achieved an increase in enrichment of the label+ cell fraction with increasing outlet numbers in the direction of the magnetic field gradient (up to 10-fold). The total recovery of the ten outlet fractions was 90.0+/-7.7%. The mean MM of label+ cells increased with increasing outlet number by up to a factor of 2.3. The postulated proportionality between the number of attached magnetic beads and the number of cell surface markers was validated by comparison of MM measured by cell tracking velocimetry (CTV) with cell florescence intensity measured by flow cytometry.     

Efficient cloning of full-length cDNAs based on cDNA size fractionation

The ability to generate and obtain full-length (FL) cDNAs is of critical importance to the field of genomics. Most cDNAs in a traditional cDNA library lack the initiating 5' ATG, making it difficult to obtain a FL clone. We report here on an improved protocol for the preparation of FL enriched cDNA libraries. We demonstrate that if good quality RNA is used in the cDNA synthesis, high-quality, FL cDNA can be generated for messages upward of 7 kb. In addition, we demonstrate the utility of size fractionation as a means to produce libraries containing a high percentage of initiating 5' ATG containing clones with insert sizes greater than 4 kb. The method is simple, cost efficient, and can be performed in most laboratories equipped to perform molecular biology. Lastly, the novel methodologies used in the analysis of the cDNA and library should prove useful to others working to create high-quality cDNA libraries.     

Analysis of membrane protein complexes by blue native PAGE

Blue native polyacryamide gel electrophoresis is a special case of native electrophoresis for high resolution separation of enzymatically active protein complexes from tissue homogenates and cell fractions. The method is powerful between 10 and 10,000 kDa. Also membrane protein complexes are separated well after solubilization of complexes with mild neutral detergents. The separation principle relies on binding of Coomassie blue G250 which provides negative charges to the surface of the protein. During migration to the anode, protein complexes are separated according to molecular mass and/or size and high resolution is obtained by the decreasing pore size of a polyacrylamide gradient gel. The principles of 2-dimensional blue native sodium dodecyl sulfate polyacrylamide gel electrophoresis are presented here together with a practical step-by-step guide to performing the method in the laboratory.     

The bioactivity and fractionation of peptide hydrolysates in cultures of CHO cells

Peptide hydrolysate supplements in mammalian cell cultures provide enhanced growth and productivity. The objective of this study was to compare the bioactivity of ten different commercially available hydrolysates from plant, microbial, and animal sources. The peptide hydrolysates were tested as supplements to cultures of Chinese hamster ovary (CHO) cells that produce human beta interferon (β‐IFN). A soy hydrolysate was shown to support high cell growth but not protein productivity compared to an animal component hydrolysate (Primatone RL). On the other hand, a yeast hydrolysate showed lower cell growth, but comparable productivity of the recombinant protein. Glycosylation analysis showed that the glycan profile of β‐IFN produced in yeast hydrolysate supplemented cultures was equivalent to that from Primatone RL‐supplemented cultures. Fractionation of the yeast hydrolysate and Primatone RL produced a similar protein‐assayed pattern except for one extra peak at around 1 kDa in the Primatone RL profile. A fraction taken at a molecular weight range of 1.5–1.7 kDa showed the highest growth promoting activity in both samples. However, four other fractions in yeast hydrolysate and two in Primatone RL at lower molecular weights showed some growth promoting activity. In conclusion, the yeast hydrolysates provided a good alternative to the animal sourced Primatone RL for high productivity of β‐IFN from CHO cells with equivalent glycosylation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:584–593, 2014     

Membrane fractionation based on functional composition: Evidence for membrane domains

This study provides evidence for the organization of a bacterial cytoplasmic membrane in functional domains. The cytoplasmic membrane ofRhodospirillum rubrum was fractionated on the basis of affinity for the -lactam agent 6-aminopenicillanic acid. Cytoplasmic membrane components were found to be differentially localized in membrane subpopulations; this implies that these membranes arose from distinct domains in the laterally differentiated cytoplasmic membrane.     

pI-based fractionation of serum proteomes versus anion exchange after enhancement of low-abundance proteins by means of peptide libraries

The pre-treatment of biological extracts with the aim of detecting very low-abundance proteins generates complexity requiring a proper fractionation. Therefore the success of identifying all newly detectable species depends on the selected fractionation methods.In this context and starting from a human serum, where the dynamic concentration range was reduced by means of a preliminary treatment with a combinatorial hexapeptide ligand library, we fractionated the sample using a novel method based on the differences in isoelectric points of proteins by means of Solid-State Buffers (SSB) associated with cation exchangers. The number of fractions was limited to four and was compared to a classical anion exchange method generating the same number of fractions.What was observed is that when using SSB technology the protein redundancy between fractions was significantly reduced compared to ion exchange fractionation allowing thus a better detection of novel species. The analysis of trypsinized protein fractions by nanoLC-MS/MS confirmed that the SSB technology used is more discriminant than anion exchange chromatography fractionation.A sample fractionation by SSB after the reduction of dynamic concentration range can be accomplished without either adjustment of pH and ionic strength or protein concentration and cleanup. Both advantages over either classical chromatography or isoelectric fractionations allow approaching the discovery of markers of interest under easier conditions applicable in a variety of fields of investigation.     

Proteomics of glycoproteins based on affinity selection of glycopeptides from tryptic digests

Identification of glycoproteins in complex mixtures derived from either human blood serum or a cancer cell line was achieved in a process involving the steps of (1) reduction and alkylation, (2) proteolysis of all proteins in the mixture with trypsin, (3) affinity chromatographic selection of the glycopeptides with an immobilized lectin, (4) direct transfer of the glycopeptide fraction to a reversed-phase liquid chromatography (RPLC) column and further fractionation by gradient elution, (5) matrix-assisted laser desorption ionization mass spectrometry of individual fractions collected from the RPLC column, and (6) peptide identification based on a database search. The types of glycoproteins analyzed were; (1) N-type glycoproteins of known primary structure, (2) N-type glycoproteins of unknown structure, and (3) O-type glycoproteins glycosylated with a single N-acetylglucosamine. Identification of peptides from complex mixtures was greatly facilitated by either C-terminal sequencing with a carboxypeptidase mixture or by comparing chromatographic behavior and mass to standards, as in the case of a known protein. In addition, deglycosylation of peptides with N glycosidase F was necessary to identify N-type glycoproteins of unknown structure. The strength of this approach is that it is fast and targets specific molecular species or classes of glycoproteins for identification. The weakness is that it does not discriminate between glycoforms.