A novel approach for protein identification from complex cell proteome using modified peptide mass fingerprinting algorithm

A unique peptide based search algorithm for identification of protein mixture using PMF is proposed. The proposed search algorithm utilizes binary search and heapsort programs to generate frequency chart depicting the unique peptides corresponding to all proteins in a proteome. The use of binary search program significantly reduces the time for frequency chart preparation to ～2 s for a proteome comprising ～23 000 proteins. The algorithm was applied to a three‐protein mixture identification, host cell protein (HCP) analysis, and a simulation‐generated data set. It was found that the algorithm could identify at least one unique peptide of a protein even in the presence of fourfold higher concentration of another protein. In addition, two HCPs that are known to be difficult to remove were missed by MS/MS approach and were exclusively identified using the presented algorithm. Thus, the proposed algorithm when used along with standard proteomic approaches present avenues for enhanced protein identification efficiency, particularly for applications such as HCP analysis in biopharmaceutical research, where identification of low‐abundance proteins are generally not achieved due to dynamic range limitations between the target product and HCPs.     

How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?

Two‐dimensional gel electrophoresis (2DE) in proteomics is traditionally assumed to contain only one or two proteins in each 2DE spot. However, 2DE resolution is being complemented by the rapid development of high sensitivity mass spectrometers. Here we compared MALDI‐MS, LC‐Q‐TOF MS and LC‐Orbitrap Velos MS for the identification of proteins within one spot. With LC‐Orbitrap Velos MS each Coomassie Blue‐stained 2DE spot contained an average of at least 42 and 63 proteins/spot in an analysis of a human glioblastoma proteome and a human pituitary adenoma proteome, respectively, if a single gel spot was analyzed. If a pool of three matched gel spots was analyzed this number further increased up to an average of 230 and 118 proteins/spot for glioblastoma and pituitary adenoma proteome, respectively. Multiple proteins per spot confirm the necessity of isotopic labeling in large‐scale quantification of different protein species in a proteome. Furthermore, a protein abundance analysis revealed that most of the identified proteins in each analyzed 2DE spot were low‐abundance proteins. Many proteins were present in several of the analyzed spots showing the ability of 2DE‐MS to separate at the protein species level. Therefore, 2DE coupled with high‐sensitivity LC‐MS has a clearly higher sensitivity as expected until now to detect, identify and quantify low abundance proteins in a complex human proteome with an estimated resolution of about 500 000 protein species. This clearly exceeds the resolution power of bottom‐up LC‐MS investigations.     

Analysis of rat testicular proteome following 30‐day exposure to 900 MHz electromagnetic field radiation

The use of electromagnetic field (EMF) generating apparatuses such as cell phones is increasing, and has caused an interest in the investigations of its effects on human health. We analyzed proteome in preparations from the whole testis in adult male Sprague‐Dawley rats that were exposed to 900 MHz EMF radiation for 1, 2, or 4 h/day for 30 consecutive days, simulating a range of possible human cell phone use. Subjects were sacrificed immediately after the end of the experiment and testes fractions were solubilized and separated via high‐resolution 2D electrophoresis, and gel patterns were scanned, digitized, and processed. Thirteen proteins, which were found only in sham or in exposure groups, were identified by MALDI‐TOF/TOF‐MS. Among them, heat shock proteins, superoxide dismutase, peroxiredoxin‐1, and other proteins related to misfolding of proteins and/or stress were identified. These results demonstrate significant effects of radio frequency modulated EMFs exposure on proteome, particularly in protein species in the rodent testis, and suggest that a 30‐day exposure to EMF radiation induces nonthermal stress in testicular tissue. The functional implication of the identified proteins was discussed.     

NSI and NSMT: usages of MS/MS fragment ion intensity for sensitive differential proteome detection and accurate protein fold change calculation in relative label-free proteome quantification

Although widely applied in the label-free quantification of proteomics, spectral count (SC)-based abundance measurements suffer from the narrow dynamic range of attainable ratios, leading to the serious underestimation of true protein abundance fold changes, especially when studying biological samples that exhibit very large fold changes in protein expression. MS/MS fragment ion intensity, as an alternative to SC, has recently gained acceptance as the abundance feature of protein in label-free proteomic studies. Herein, we implemented two formats of MS/MS fragment ion intensity, Spectral Index (SI) and Summed MS/MS TIC (SMT), to alleviate this particular deficiency arising from SC. Both were in forms of replacing SC in the Normalized Spectral Abundance Factor (NSAF) formula, resulting in two algorithms, abbreviated as NSI and NSMT, respectively. The necessity of the normalization process was validated using a publicly available dataset. Furthermore, when applied to another well characterized benchmark dataset, both NSI and NSMT showed improved overall accuracy over NSAF for the relative quantification of proteomes. Hereinto, NSI enabled the sensitive detection of differentially expressed proteins, while NSMT ensured accurate calculation for protein abundance fold change. Therefore, the selective use of both algorithms might facilitate the screening and quantification of potential biomarkers on the proteome scale.     

Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionation

Despite its excellent resolving power, 2-DE is of limited use when analyzing cellular proteomes, especially in differential expression studies. Frequently, fewer than 2000 protein spots are detected on a single 2-D gel (a fraction of the total proteome) regardless of the gel platform, sample, or detection method used. This is due to the vast number of proteins expressed and their equally vast dynamic range. To exploit 2-DE unique ability as both an analytical and a preparative tool, the significant sample prefractionation is necessary. We have used solution isoelectric focusing (sIEF) via the ZOOM IEF Fractionator (Invitrogen) to generate sample fractions from complex bacterial lysates, followed by parallel 2-DE, using narrow-range IPG strips that bracket the sIEF fractions. The net result of this process is a significant enrichment of the bacterial proteome resolved on multiple 2-D gels. After prefractionation, we detected 5525 spots, an approximate 3.5-fold increase over the 1577 spots detected in an unfractionated gel. We concluded that sIEF is an effective means of prefractionation to increase depth of field and improve the analysis of low-abundance proteins.     

Spot volume vs. amount of protein loaded onto a gel: a detailed,statistical comparison of two gel electrophoresis systems

The long-term goal of this research program is to clarify the molecular mechanisms that participate in the formation of human pituitary macroadenomas. One approach to that goal is to characterize the differentially expressed proteins that are found by a comparison of the proteomes of control pituitary vs. macroadenoma tissues. In order to accurately perform a comparative proteomics study, based on the combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and PDQuest 2-D analysis software, a reproducible 2-DE separation system with a wide linear dynamic measure range is needed. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180 x 245 x 0.5 mm); a typical vertical system is the Dodeca system that analyzes up to 12 gels at a time on a single-concentration gel (190 x 205 x 1.0 mm). We have evaluated (Zhan and Desiderio, Electrophoresis 2003, 24, 1834-1846) the spatial and quantitative reproducibility of the two second-dimensional gel systems to separate a human pituitary proteome; that study showed a higher reproducibility for the Dodeca gel system. This present study investigated the relationship between the spot volume and the amount of protein loaded onto the gel for those two 2-D systems. The results demonstrated that the Dodeca gel system provides a wider linear dynamic range to measure the changes in the protein abundance in pituitary proteome.     

High‐throughput negative detection of SDS‐PAGE separated proteins and its application for proteomics

A negative detection method for proteins on SDS‐PAGE is described. In this method, Eosin Y (EY) was selectively precipitated in the gel background, which is absent from those zones where proteins are located through the formation of a stable water‐soluble protein–dye complex. Negative staining of proteins using EY, allows high‐sensitivity, low‐cost, and simple protocol. The new described method takes less than an hour to complete all the protocol, with a detection limit of 0.5 ng of single protein band. Comparing with imidazole‐zinc negative stain, EY dye provides broader linear dynamic range, higher sensitivity and reproducibility, and better obvious contrast between the protein bands or spots and background. Furthermore, the novel technique developed here presented a real practical method for simultaneous processing of multiple gels, which makes it possible to perform high‐throughput staining for proteome research. Additionally, we have also compared the influence of staining method on the quality of mass spectra by PMF.     

Fluorogenic Strain‐Promoted Alkyne–Diazo Cycloadditions

Fluorogenic reactions, in which non‐ or weakly fluorescent reagents produce highly fluorescent products, are attractive for detecting a broad range of compounds in the fields of bioconjugation and material sciences. Herein, we report that a dibenzocyclooctyne derivative modified with a cyclopropenone moiety (Fl‐DIBO) can undergo fast strain‐promoted cycloaddition reactions under catalyst‐free conditions with azides, nitrones, nitrile oxides, as well as mono‐ and disubstituted diazo‐derivatives. Although the reaction with nitrile oxides, nitrones, and disubstituted diazo compounds gave cycloadducts with low quantum yield, monosubstituted diazo reagents produced 1H‐pyrazole derivatives that exhibited an approximately 160‐fold fluorescence enhancement over Fl‐DIBO combined with a greater than 10 000‐fold increase in brightness. Concluding from quantum chemical calculations, fluorescence quenching of 3H‐pyrazoles, which are formed by reaction with disubstituted diazo‐derivatives, is likely due to the presence of energetically low‐lying (n,π*) states. The fluorogenic probe Fl‐DIBO was successfully employed for the labeling of diazo‐tagged proteins without detectable background signal. Diazo‐derivatives are emerging as attractive reporters for the labeling of biomolecules, and the studies presented herein demonstrate that Fl‐DIBO can be employed for visualizing such biomolecules without the need for probe washout.     

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).     

Dried polyacrylamide gel absorption: a method for efficient elimination of the interferences from SDS-solubilized protein samples in mass spectrometry-based proteome analysis

Sample preparation holds an important place in MS-based proteome analysis. For effective proteolysis and MS analysis, it is essential to eliminate the interferences while extracting the analytes of interest from complex mixtures. To address this, herein we describe a new dried polyacrylamide gel absorption method. In this method, the protein sample prepared using high concentration of SDS was directly and completely absorbed by vacuum-dried polyacrylamide gel, and then the interfering substances including SDS and some other salts were efficiently removed by in-gel washing steps while retaining the denatured proteins in the gel, thus offering a clean environment amenable to downstream buffer exchange, proteolytic digestion and digest recovery, etc. In combination with in-gel digestion and LC-MS/MS, the newly developed method was applied to the proteome analyses of membrane-enriched fraction and whole tissue homogenate. It was demonstrated that the method is suitable for the analysis of a complex biological sample and can be widely used for sample cleanup in shotgun proteome analyses.     

A novel subfractionation approach for mitochondrial proteins: a three-dimensional mitochondrial proteome map

As mitochondria play critical roles in both cell life and cell death, there is great interest in obtaining a human mitochondrial proteome map. Such a map could potentially be useful in diagnosing diseases, identifying targets for drug therapy, and in screening for unwanted drug side effects. In this paper, we present a novel approach to obtaining a human mitochondrial proteome map that combines sucrose gradient centrifugation with standard two-dimensional gel electrophoresis. The resulting three-dimensional separation of proteins allows us to address some of the problems encountered during previous attempts to obtain mitochondrial proteome maps such as resolution of proteins and solubility of hydrophobic proteins during isoelectric focusing. In addition, we show that this new approach provides functional information about protein complexes within the organelle that is not obtained with two-dimensional gel electrophoresis of whole mitochondria.     

Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization

Nuclear magnetic resonance (NMR) spectroscopy has the intrinsic capabilities to investigate proteins in native environments. In general, however, NMR relies on non‐natural protein purity and concentration to increase the desired signal over the background. We here report on the efficient and specific hyperpolarization of low amounts of a target protein in a large isotope‐labeled background by combining dynamic nuclear polarization (DNP) and the selectivity of protein interactions. Using a biradical‐labeled ligand, we were able to direct the hyperpolarization to the protein of interest, maintaining comparable signal enhancement with about 400‐fold less radicals than conventionally used. We could selectively filter out our target protein directly from crude cell lysate obtained from only 8 mL of fully isotope‐enriched cell culture. Our approach offers effective means to study proteins with atomic resolution in increasingly native concentrations and environments.     

Combination of affinity depletion of abundant proteins and reversed-phase fractionation in proteomic analysis of human plasma/serum

The tremendous complexity of the serum and plasma proteome presents extreme analytical challenges in comprehensive analysis due to the wide dynamic range of protein concentrations. Therefore, robust sample preparation methods remain one of the important steps in the proteome characterization workflow. We present the results on a new column for the specific depletion of 14 high-abundant proteins from human serum and plasma and the subsequent reversed-phase fractionation of the flow-through proteins. Analysis of tryptic peptides was accomplished with microfluidic HPLC-Chip/MS system. Results indicate that high-abundant protein depletion combined with RP fractionation of plasma showed an improved dynamic range for proteomic analysis and enabled the identification of low-abundant plasma proteins.     

A magnetic bead‐based serum proteomic fingerprinting method for parallel analytical analysis and micropreparative purification

ProteinChip surface‐enhanced laser desorption/ionization technology and magnetic beads‐based ClinProt system are commonly used for semi‐quantitative profiling of plasma proteome in biomarker discovery. Unfortunately, the proteins/peptides detected by MS are non‐recoverable. To obtain the protein identity of a MS peak, additional time‐consuming and material‐consuming purification steps have to be done. In this study, we developed a magnetic beads‐based proteomic fingerprinting method that allowed semi‐quantitative proteomic profiling and micropreparative purification of the profiled proteins in parallel. The use of different chromatographic magnetic beads allowed us to obtain different proteomic profiles, which were comparable to those obtained by the ProteinChip surface‐enhanced laser desorption/ionization technology. Our assays were semi‐quantitative. The normalized peak intensity was proportional to concentration measured by immunoassay. Both intra‐assay and inter‐assay coefficients of variation of the normalized peak intensities were in the range of 4–30%. Our method only required 2 μL of serum or plasma for generating enough proteins for semi‐quantitative profiling by MALDI‐TOF‐MS as well as for gel electrophoresis and subsequent protein identification. The protein peaks and corresponding gel spots could be easily matched by comparing their intensities and masses. Because of its high efficiency and reproducibility, our method has great potentials in clinical research, especially in biomarker discovery.     

Surface potential mapping of dispersed proteins

We describe a method for detecting proteins after transfer to PVDF membranes, based on the surface potential attributed to each protein. Proteins separated by classical two-dimensional polyacrylamide gel electrophoresis could be detected by scanning the membrane surface with a vibrating capacitor (also called a Kelvin probe) on the basis of differences between their surface potential and that of the membrane. Coupled to colloidal gold staining, the technique enables detection of proteins previously undetectable by classical staining methods. Plotting variations of the surface potential in two dimensions visualizes proteins which migrate close together. Finally, we demonstrate that the Kelvin probe detects proteins over a concentration range from micro to sub-nanogram with increased sensitivity at lower concentrations, and unlike other methods, appears to be similar for all proteins tested so far. The method described is fast, reliable, and it can be automated for high throughput.     

Precursor ion scans for the targeted detection of stable‐isotope‐labeled peptides

Stable isotope labels are routinely introduced into proteomes for quantification purposes. Full labeling of cells in varying biological states, followed by sample mixing, fractionation and intensive data acquisition, is used to obtain accurate large‐scale quantification of total protein levels. However, biological processes often affect only a small group of proteins for a short time, resulting in changes that are difficult to detect against the total proteome background. An alternative approach could be the targeted analysis of the proteins synthesized in response to a given biological stimulus. Such proteins can be pulse‐labeled with a stable isotope by metabolic incorporation of ‘heavy’ amino acids. In this study we investigated the specific detection and identification of labeled proteins using acquisition methods based on Precursor Ion Scans (PIS) on a triple‐quadrupole ion trap mass spectrometer. PIS‐based methods were set to detect unique immonium ions originating from labeled peptides. Different labels and methods were tested in standard mixtures to optimize performance. We showed that, in comparison with an untargeted analysis on the same instrument, the approach allowed a several‐fold increase in the specificity of detection of labeled proteins over unlabeled ones. The technique was applied to the identification of proteins secreted by human cells into growth media containing bovine serum proteins, allowing the preferential detection of labeled cellular proteins over unlabeled bovine ones. However, compared with untargeted acquisitions on two different instruments, the PIS‐based strategy showed some limitations in sensitivity. We discuss possible perspectives of the technique. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

A comparative analysis of human plasma and serum proteins by combining native PAGE,whole‐gel slicing and quantitative LC‐MS/MS: Utilizing native MS‐electropherograms in proteomic analysis for discovering structure and interaction‐correlated differences

MS identification has long been used for PAGE‐separated protein bands, but global and systematic quantitation utilizing MS after PAGE has remained rare and not been reported for native PAGE. Here we reported on a new method combining native PAGE, whole‐gel slicing and quantitative LC‐MS/MS, aiming at comparative analysis on not only abundance, but also structures and interactions of proteins. A pair of human plasma and serum samples were used as test samples and separated on a native PAGE gel. Six lanes of each sample were cut, each lane was further sliced into thirty‐five 1.1 mm × 1.1 mm squares and all the squares were subjected to standardized procedures of in‐gel digestion and quantitative LC‐MS/MS. The results comprised 958 data rows that each contained abundance values of a protein detected in one square in eleven gel lanes (one plasma lane excluded). The data were evaluated to have satisfactory reproducibility of assignment and quantitation. Totally 315 proteins were assigned, with each protein assigned in 1–28 squares. The abundance distributions in the plasma and serum gel lanes were reconstructed for each protein, named as “native MS‐electropherograms”. Comparison of the electropherograms revealed significant plasma‐versus‐serum differences on 33 proteins in 87 squares (fold difference > 2 or < 0.5, p < 0.05). Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation, complement and wound healing processes. We expect this method would be useful to provide more comprehensive information in comparative proteomic analysis, on both quantities and structures/interactions.     

Over 10 000 Peptide Identifications from the HeLa Proteome by Using Single‐Shot Capillary Zone Electrophoresis Combined with Tandem Mass Spectrometry

Capillary zone electrophoresis (CZE)–tandem mass spectrometry (MS/MS) has recently attracted attention as a tool for shotgun proteomics. However, its performance for this analysis has so far fallen far below that of reversed‐phase liquid chromatography (RPLC)–MS/MS. The use of a CZE method with a wide separation window (up to 90 min) and high peak capacity (ca. 300) is reported. This method was coupled to an Orbitrap Fusion mass spectrometer through an electrokinetically pumped sheath‐flow interface for the analysis of complex proteome digests. Single‐shot CZE–MS/MS lead to the identification of over 10 000 peptides and 2100 proteins from a HeLa cell proteome digest in approximately 100 min. This performance is nearly an order of magnitude better than earlier CZE studies and is within a factor of two to four of the state‐of‐the‐art nano ultrahigh‐pressure LC system.     

Improved dynamic range of protein quantification in silver‐stained gels by modelling gel images over time

Silver staining is a commonly used protein stain to visualise proteins separated by 2‐DE. Despite this, the technique suffers from a limited dynamic range, making the simultaneous quantification of high‐ and low‐abundant proteins difficult. In this paper we take advantage of the fact that silver staining is not an end‐point stain by photographing the gels during development. This procedure provides information about the change in measured absorbance for each pixel in the protein spots on the gel. The maximum rate of change was found to be correlated with the amount of applied protein, providing a new way of estimating protein amount in 2‐DE gels. We observed an improvement in the dynamic range of silver staining by up to two orders of magnitude.