Use of DNA ladders for reproducible protein fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for quantitative proteomics

In proteomics, one-dimensional (1D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is widely used for protein fractionation prior to mass spectrometric analysis to enhance the dynamic range of analysis and to improve the identification of low-abundance proteins. Such protein prefractionation works well for quantitation strategies if the proteins are labeled prior to separation. However, because of the poor reproducibility of cutting gel slices, especially when small amounts of samples are analyzed, its application in label-free and peptide-labeling quantitative proteomics methods has been greatly limited. To overcome this limitation, we developed a new strategy in which a DNA ladder is mixed with the protein sample before PAGE separation. After PAGE separation, the DNA ladder is stained to allow for easy, precise, and reproducible gel cutting. To this end, a novel visible DNA-staining method was developed. This staining method is fast, sensitive, and compatible with mass spectrometry. To evaluate the reproducibility of DNA-ladder-assisted gel cutting for quantitative protein fractionation, we used stable isotope labeling with amino acids in cell culture (SILAC). Our results show that the quantitative error associated with fractionation can be minimized using the DNA-assisted fractionation and multiple replicates of gel cutting. In conclusion, 1D PAGE fractionation in combination with DNA ladders can be used for label-free comparative proteomics without compromising quantitation.     

Application of a peptide-based PF2D platform for quantitative proteomics in disease biomarker discovery

A peptide-based 2-D liquid phase fractionation (PF2D) system was used in a quantitative proteomic analysis of hepatocellular carcinoma. 2-D liquid maps of peptide specimens showed better resolution than those of proteins, leading to the identification of differentially expressed proteins. Peptide-based PF2D gave well-matched theoretical and experimental pI values and was proven to be a very efficient and versatile analytical tool for both large-scale profiling and quantification of phosphoproteins in disease biomarker discovery.     

Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes

In-depth analysis of the serum and plasma proteomes by mass spectrometry is challenged by the vast dynamic range of protein abundance and substantial complexity. There is merit in reducing complexity through fractionation to facilitate mass spectrometry analysis of low-abundance proteins. However, fractionation reduces throughput and has the potential of diluting individual proteins or inducing their loss. Here, we have investigated the contribution of extensive fractionation of intact proteins to depth of analysis. Pooled serum depleted of abundant proteins was fractionated by an orthogonal two-dimensional system consisting of anion-exchange and reversed-phase chromatography. The resulting protein fractions were aliquotted; one aliquot was analyzed by shotgun LC-MS/MS, and another was further resolved into protein bands in a third dimension using SDS-PAGE. Individual gel bands were excised and subjected to in situ digestion and mass spectrometry. We demonstrate that increased fractionation results in increased depth of analysis based on total number of proteins identified in serum and based on representation in individual fractions of specific proteins identified in gel bands following a third-dimension SDS gel analysis. An intact protein analysis system (IPAS) based on a two-dimensional plasma fractionation schema was implemented that resulted in identification of 1662 proteins with high confidence with representation of protein isoforms that differed in their chromatographic mobility. Further increase in depth of analysis was accomplished by repeat analysis of aliquots from the same set of two-dimensional fractions resulting in overall identification of 2254 proteins. We conclude that substantial depth of analysis of proteins from milliliter quantities of serum or plasma and detection of isoforms are achieved with depletion of abundant proteins followed by two-dimensional protein fractionation and MS analysis of individual fractions.     

A proteomic approach for evaluating the cell response to a novel histone deacetylase inhibitor in colon cancer cells

Epigenetic inactivation of gene expression is a general phenomenon associated with malignant transformation. Recently, we have found that a novel series of histone deacetylases (HDAC) inhibitors exhibit a broad-spectrum inhibition profile characterized by a marked effect on acetylation of histone and non-histone proteins. RC307, a representative compound of this series, caused a growth-inhibitory effect in colon carcinoma cells HCT116 associated with G2 accumulation and induction of apoptosis. The present study was designed to investigate the effect of RC307 on protein expressions in the HCT116 cells following treatment with cytotoxic drug concentrations. HCT116 cells were cultured in the absence or presence of RC307 and total cell lysates, as well as nuclear proteins, were extracted. The protein samples were then subjected to two-dimensional polyacrylamide gel electrophoresis, and the 2D gel images were compared to discover the protein changes caused by RC307 treatment. A total of 48 and 46 different spots were found to be modulated by RC307 in total lysates and nuclear proteome of HCT116 cell line. The modulated proteins were identified by tandem mass spectrometry. We found that RC307 exposure modulates proteins that are involved in proliferation, cell cycle regulation, apoptosis, gene expression, as well as chromatin and cytoskeleton organization.     

Quantitative proteome analysis using isotope-coded affinity tags and mass spectrometry

A main objective of proteomics research is to systematically identify and quantify proteins in a given proteome (cells, subcellular fractions, protein complexes, tissues or body fluids). Protein labeling with isotope-coded affinity tags (ICAT) followed by tandem mass spectrometry allows sequence identification and accurate quantification of proteins in complex mixtures, and has been applied to the analysis of global protein expression changes, protein changes in subcellular fractions, components of protein complexes, protein secretion and body fluids. This protocol describes protein-sample labeling with ICAT reagents, chromatographic fractionation of the ICAT-labeled tryptic peptides, and protein identification and quantification using tandem mass spectrometry. The method is suitable for both large-scale analysis of complex samples including whole proteomes and small-scale analysis of subproteomes, and allows quantitative analysis of proteins, including those that are difficult to analyze by gel-based proteomics technology.     

Evaluation of reproducibility of protein identification results after multidimensional human serum protein separation

We developed a gel- and label-free proteomics platform for comparative studies of human serum. The method involves the depletion of the six most abundant proteins, protein fractionation by Off-Gel IEF and RP-HPLC, followed by tryptic digestion, LC-MS/MS, protein identification, and relative quantification using probabilistic peptide match score summation (PMSS). We evaluated performance and reproducibility of the complete platform and the individual dimensions, by using chromatograms of the RP-HPLC runs, PMSS based abundance scores and abundance distributions as objective endpoints. We were interested if a relationship exists between the quantity ratio and the PMSS score ratio. The complete analysis was performed four times with two sets of serum samples containing different concentrations of spiked bovine beta-lactoglobulin (0.1 and 0.3%, w/w). The two concentrations resulted in significantly differing PMSS scores when compared to the variability in PMSS scores of all other protein identifications. We identified 196 proteins, of which 116 were identified four times in corresponding fractions whereof 73 qualified for relative quantification. Finally, we characterized the PMSS based protein abundance distributions with respect to the two dimensions of fractionation and discussed some interesting patterns representing discrete isoforms. We conclude that combination of Off-Gel electrophoresis (OGE) and HPLC is a reproducible protein fractionation technique, that PMSS is applicable for relative quantification, that the number of quantifiable proteins is always smaller than the number of identified proteins and that reproducibility of protein identifications should supplement probabilistic acceptance criteria.     

Large scale protein profiling by combination of protein fractionation and multidimensional protein identification technology (MudPIT)

In the past decade, shotgun proteomic analysis has been utilized extensively to answer complex biological questions. New challenges arise in large scale proteomic profiling when dealing with complex biological mixtures such as the mammalian cell lysate. In this study, we explored the approach of protein separation prior to the shotgun multidimensional protein identification technology (MudPIT) analysis. We fractionated the mammalian cancer cell lysate using the PF 2D ProteomeLab system and analyzed the distribution of molecular weight, isoelectric point, and cellular localization of the eluted proteins. As a result, we were able to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins with lower abundance in the complex protein mixture.     

Label-free quantification and shotgun analysis of complex proteomes by one-dimensional SDS-PAGE/NanoLC-MS: evaluation for the large scale analysis of inflammatory human endothelial cells

To perform differential studies of complex protein mixtures, strategies for reproducible and accurate quantification are needed. Here, we evaluated a quantitative proteomic workflow based on nanoLC-MS/MS analysis on an LTQ-Orbitrap-VELOS mass spectrometer and label-free quantification using the MFPaQ software. In such label-free quantitative studies, a compromise has to be found between two requirements: repeatability of sample processing and MS measurements, allowing an accurate quantification, and high proteomic coverage of the sample, allowing quantification of minor species. The latter is generally achieved through sample fractionation, which may induce experimental bias during the label-free comparison of samples processed, and analyzed independently. In this work, we wanted to evaluate the performances of MS intensity-based label-free quantification when a complex protein sample is fractionated by one-dimensional SDS-PAGE. We first tested the efficiency of the analysis without protein fractionation and could achieve quite good quantitative repeatability in single-run analysis (median coefficient of variation of 5%, 99% proteins with coefficient of variation <48%). We show that sample fractionation by one-dimensional SDS-PAGE is associated with a moderate decrease of quantitative measurement repeatability while largely improving the depth of proteomic coverage. We then applied the method for a large scale proteomic study of the human endothelial cell response to inflammatory cytokines, such as TNFα, interferon γ, and IL1β, which allowed us to finely decipher at the proteomic level the biological pathways involved in endothelial cell response to proinflammatory cytokines.     

Characterization and Quantification of Intact 26S Proteasome Proteins by Real-Time Measurement of Intrinsic Fluorescence Prior to Top-down Mass Spectrometry

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.     

Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity

Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest—in this case CB2—but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.     

The Effect of Peptide Identification Search Algorithms on MS2-Based Label-Free Protein Quantification

Abstract Several approaches exist for the quantification of proteins in complex samples processed by liquid chromatography-mass spectrometry followed by fragmentation analysis (MS2). One of these approaches is label-free MS2-based quantification, which takes advantage of the information computed from MS2 spectrum observations to estimate the abundance of a protein in a sample. As a first step in this approach, fragmentation spectra are typically matched to the peptides that generated them by a search algorithm. Because different search algorithms identify overlapping but non-identical sets of peptides, here we investigate whether these differences in peptide identification have an impact on the quantification of the proteins in the sample. We therefore evaluated the effect of using different search algorithms by examining the reproducibility of protein quantification in technical repeat measurements of the same sample. From our results, it is clear that a search engine effect does exist for MS2-based label-free protein quantification methods. As a general conclusion, it is recommended to address the overall possibility of search engine-induced bias in the protein quantification results of label-free MS2-based methods by performing the analysis with two or more distinct search engines.     

Characterization of dihydroartemisinin-resistant colon carcinoma HCT116/R cell line

Dihydroartemisinin (DHA) is an important artemisinin derivative and presents profound anti-tumor potential. A DHA-resistant cell line named HCT116/R derived from colon carcinoma cell line HCT116 was established in our previous study. Herein, we found that HCT116/R cells were much more resistant to DHA- or artesunate-induced proliferation inhibition and more tolerant to DHA-induced cell cycle arrest and apoptosis compared with those of the parent HCT116 cells. The protein levels of P-glycoprotein and MDR-associated protein 1 and the accumulation of doxorubicin in cells were similar in both cell lines. Moreover, HCT116/R cells were still sensitive to camptothecin- and doxorubicin-induced cell growth inhibition. To further explore the characterization of HCT116/R cell line, a proteomic study employing two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was performed. Eight different expressed proteins between the two cell lines were identified including some heat shock proteins, annexins, etc. This study not only indicates that exposure to DHA may not induce a tumor multi-drug-resistant phenotype but also affords new clues for the further investigation of the anti-cancer mechanisms of DHA and other artemisinin derivatives.     

Computational methods for the comparative quantification of proteins in label-free LCn-MS experiments

Liquid chromatography (LC) coupled to electrospray mass spectrometry (MS) is well established in high-throughput proteomics. The technology enables rapid identification of large numbers of proteins in a relatively short time. Comparative quantification of identified proteins from different samples is often regarded as the next step in proteomics experiments enabling the comparison of protein expression in different proteomes. Differential labeling of samples using stable isotope incorporation or conjugation is commonly used to compare protein levels between samples but these procedures are difficult to carry out in the laboratory and for large numbers of samples. Recently, comparative quantification of label-free LC(n)-MS proteomics data has emerged as an alternative approach. In this review, we discuss different computational approaches for extracting comparative quantitative information from label-free LC(n)-MS proteomics data. The procedure for computationally recovering the quantitative information is described. Furthermore, statistical tests used to evaluate the relevance of results will also be discussed.     

A review on recent developments in mass spectrometry instrumentation and quantitative tools advancing bacterial proteomics

Proteomics has evolved substantially since its early days, some 20 years ago. In this mini-review, we aim to provide an overview of general methodologies and more recent developments in mass spectrometric approaches used for relative and absolute quantitation of proteins. Enhancement of sensitivity of the mass spectrometers as well as improved sample preparation and protein fractionation methods are resulting in a more comprehensive analysis of proteomes. We also document some upcoming trends for quantitative proteomics such as the use of label-free quantification methods. Hopefully, microbiologists will continue to explore proteomics as a tool in their research to understand the adaptation of microorganisms to their ever changing environment. We encourage them to incorporate some of the described new developments in mass spectrometry to facilitate their analyses and improve the general knowledge of the fascinating world of microorganisms.     

Mass Spectrometry-Based Approaches Toward Absolute Quantitative Proteomics

Mass spectrometry has served as a major tool for the discipline of proteomics to catalogue proteins in an unprecedented scale. With chemical and metabolic techniques for stable isotope labeling developed over the past decade, it is now routinely used as a method for relative quantification to provide valuable information on alteration of protein abundance in a proteome-wide scale. More recently, absolute or stoichiometric quantification of proteome is becoming feasible, in particular, with the development of strategies with isotope-labeled standards composed of concatenated peptides. On the other hand, remarkable progress has been also made in label-free quantification methods based on the number of identified peptides. Here we review these mass spectrometry-based approaches for absolute quantification of proteome and discuss their implications.Key Words: Quantitative proteomics, mass spectrometry, absolute quantification, stable isotope labeling, label-free.     

Multiplexed complexome profiling using tandem mass tags

Complexome profiling is a rapidly spreading, powerful technique to gain insight into the nature of protein complexes. It identifies and quantifies protein complexes separated into multiple fractions of increasing molecular mass using mass spectrometry-based, label-free bottom-up proteomics. Complexome profiling enables a sophisticated and thorough characterization of the composition, molecular mass, assembly, and interactions of protein complexes. However, in practice, its application is limited by the large number of samples it generates and the related time of mass spectrometry analyses. Here, we report an improved process workflow that implements tandem mass tags for multiplexing complexome profiling. This workflow substantially reduces the number of samples and measuring time without compromising protein identification or quantification reliability. In profiles from mitochondrial fractions of cells recovering from chloramphenicol treatment, tandem mass tags-multiplexed complexome profiling exhibited migration patterns of mature ATP synthase (complex V) and assembly intermediates that were consistent in composition and abundance with profiles obtained by the label-free approach. Reporter ion quantifications of proteins and complexes unaffected by the chloramphenicol treatment presented less variation in comparison to the label-free method. Incorporation of tandem mass tags enabled an efficient and robust complexome profiling analysis and may foster broader application for protein complex profiling in biomedical research and diagnostics.     

Protein separation and characterization by np-RP-HPLC followed by intact MALDI-TOF mass spectrometry and peptide mass mapping analyses

Because of their complexity, the separation of intact proteins from complex mixtures is an important step to comparative proteomics and the identification and characterization of the proteins by mass spectrometry (MS). In the study reported, we evaluated the use of nonporous-reversed-phase (np-RP)-HPLC for intact protein separation prior to MS analyses. The separation system was characterized and compared to 1D-SDS-PAGE electrophoresis in terms of resolution and sensitivity. We demonstrate that np-RP-HPLC protein separation is highly reproducible and provides intact protein fractions which can be directly analyzed by MALDI-TOF-MS for intact molecular weight determination. An in-well digestion protocol was developed, allowing for rapid protein identification by peptide mass fingerprinting (PMF) and resulted in comparable or improved peptide recovery compared with in-gel digestion. The np-RP sensitivity of detection by UV absorbance at 214 nm for intact proteins was at the low ng level and the sensitivity of peptide analysis by MALDI-TOF-MS was in the 10-50 fmol level. A membrane protein fraction was characterized to demonstrate application of this methodology. Among the identified proteins, multiple forms of vimentin were observed. Overall, we demonstrate that np-RP-HPLC followed by MALDI-TOF-MS allows for rapid, sensitive, and reproducible protein fractionation and very specific protein characterization by integration of PMF analysis with MS intact molecular weight information.     

Integrated mass spectrometry-based analysis of plasma glycoproteins and their glycan modifications

We present a protocol for the identification of glycosylated proteins in plasma followed by elucidation of their individual glycan compositions. The study of glycoproteins by mass spectrometry is usually based on cleavage of glycans followed by separate analysis of glycans and deglycosylated proteins, which limits the ability to derive glycan compositions for individual glycoproteins. The methodology described here consists of 2D HPLC fractionation of intact proteins and liquid chromatography-multistage tandem mass spectrometry (LC-MS/MS(n)) analysis of digested protein fractions. Protein samples are separated by 1D anion-exchange chromatography (AEX) with an eight-step salt elution. Protein fractions from each of the eight AEX elution steps are transferred onto the 2D reversed-phase column to further separate proteins. A digital ion trap mass spectrometer with a wide mass range is then used for LC-MS/MS(n) analysis of intact glycopeptides from the 2D HPLC fractions. Both peptide and oligosaccharide compositions are revealed by analysis of the ion fragmentation patterns of glycopeptides with an intact glycopeptide analysis pipeline.     

Fractionation of soluble proteins in Escherichia coli using DEAE-, SP-, and phenyl sepharose chromatographies.

In an effort to simplify a complex mixture of soluble proteins from Escherichia coli, methods to fractionate the samples prior to two-dimensional (2D) gel electrophoresis were developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins from an E. coli extract with DEAE-Sepharose resulted in a threefold increase in the number of detectable 2D gel spots. These gel spots were amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. Significantly, the DEAE-Sepharose column fractionation effectively partitioned the soluble proteins from the cell extracts. Similarly, an SP-Sepharose column was used to fractionate the soluble proteins from E. coli and resulted in over a twofold increase in the number of detectable gel spots. Lastly, fractionation of the cell extract with the phenyl Sepharose column resulted in a threefold increase in the number of detectable 2D gel spots. This work describes an easy, inexpensive way to fractionate the soluble proteins in E. coli and a way to better profile the E. coli proteome.     

Quantitative analysis of membrane proteins from breast cancer cell lines BT474 and MCF7 using multistep solid phase mass tagging and 2D LC/MS

We introduce a new multistep mass tagging technique and show its utility for reducing sample complexity when coupled with two-dimensional liquid chromatography/nano-electrospray ionization ion trap mass spectrometry (2D LC/nano ESI-MS). Solid-phase mass tagging reagents were used to identify and obtain relative quantitation of membrane proteins from two established breast cancer cell lines, BT474 and MCF7. The results presented in this study show that sample complexity can be reduced with corresponding increases in protein identification and quantitation.