Peptide enrichment by microfluidic electrocapture for online analysis by electrospray mass spectrometry

Identification of peptides from a complex mixture can be difficult because of the wide concentration range and the different ionization efficiencies of peptides during analysis by electrospray ionization (ESI) mass spectrometry (MS). Preconcentration methods are necessary to allow low-abundance and low-intensity peptides to reach the ionization threshold of the mass spectrometer. Here we demonstrate peptide enrichment based on electroimmobilization. Peptides are immobilized without the use of solid support or chemical binding by application of an electric field along a microflow stream in an electrocapture cell. Once enriched/preconcentrated inside the cell, they are released by removal of the electric field and via an interface with an electrospray emitter are submitted to online mass spectrometric analysis. Tandem mass spectrometric analysis of a peptide mixture containing hemoglobin, myoglobin, bovine serum albumin (BSA), and cytochrome c was successful. Amplification factors up to 16-fold were achieved with improvement of the signal-to-noise values for the preconcentrated sample. The limit of detection for one of the preconcentrated peptides was 3.6 fmol.     

Characterization of phosphorylation sites on Tpl2 using IMAC enrichment and a linear ion trap mass spectrometer

Advances in analytical techniques, specifically in mass spectrometry, have allowed for both facile protein identification and routine sequencing of proteins at increased sensitivity levels. Protein modifications present additional challenges because they occur at low stoichiometries and often change the analytical behavior of the molecule. For example, characterization of protein phosphorylation provides crucial information to signaling processes that are often associated with disease. Research into protein phosphorylation requires inter-disciplinary co-operation involving multiple investigators with expertise in diverse scientific fields. As such, techniques must be simple, effective, and incorporate multiple checkpoints that confirm the sample contains a phosphorylated protein in order to ensure resources are conserved. In this study, tumor progression locus 2 (Tpl2), which has been implicated in cell cycle regulation and has been shown to play a significant role in critical signal transduction pathways, was transfected into 293T cells, overexpressed and isolated from the cell lysate. Isolated proteins were separated via 1D gel electrophoresis, and their phosphorylation was confirmed using phosphospecific staining. The bands were excised and subjected to tryptic digestion and immobilized metal affinity chromatography (IMAC) prior to analysis by capillary-LC-MS/MS. Three phosphorylation sites were detected on Tpl2. One site had previously been reported in the literature but had not been characterized by mass spectrometric methods until this time; two additional novel sites of phosphorylation were detected.     

Identification of okadaic acid-induced phosphorylation events by a mass spectrometry approach

Okadaic acid (OA) is a widely used small-molecule phosphatase inhibitor that is thought to selectively inhibit protein phosphatase 2A (PP2A). Multiple studies have demonstrated that PP2A activity is compromised in the brains of Alzheimer's disease patients. Thus, we set out to determine changes in phosphorylation that occur upon OA treatment of neuronal cells. Utilizing isotope-coded affinity tags and mass spectrometry analysis, we determined the relative abundance of proteins in a phosphoprotein enriched fraction from control and OA-treated primary cortical neurons. We identified many proteins whose phosphorylation state is regulated by OA, including glycogen synthase kinase 3beta, collapsin-response mediator proteins (DRP-2, DPYSL-5, and CRMP-4), and the B subunit of PP2A itself. Most interestingly, we have found that complexin 2, an important regulator of neurotransmitter release and synaptic plasticity, is phosphorylated at serine 93 upon OA treatment of neurons. This is the first report of a phosphorylation site on complexin 2.     

Development of an off-line capillary column IMAC phosphopeptide enrichment method for label-free phosphorylation relative quantification

Immobilized metal affinity chromatography (IMAC) and metal oxide type affinity chromatography (MOAC) techniques have been widely used for mass spectrometry-based phosphorylation analysis. Unlike MOAC techniques, IMAC requires rather complete removals of buffering reagents, salts and high concentrations of denaturant prior to sample loading in order for the successful enrichment of phosphopeptides. In this study, a simple off-line capillary column-based IMAC phosphopeptide enrichment method can shorten sample preparation time by eliminating the speed-vac step from the desalting process. Tryptic digest peptide samples containing 2M urea can be directly processed and the entire IMAC procedure can be completed within 6 h. When tryptic digest peptide samples prepared from mouse whole brain tissues were analyzed using our method, an average of 249 phosphoproteins and 463 unique phosphopeptides were identified from single 2-h RPLC-MS/MS analysis (~88% specificity). An additional advantage of this method is the significantly improved reproducibility of the phosphopeptide enrichment results. When four independent phosphopeptide enrichment experiments were carried out, the peak areas of phosphopeptides identified among four enrichment experiments were relatively similar (less than 16.2% relative standard dev.). Because of this increased reproducibility, relative phosphorylation quantification analysis of major phosphoproteins appears to be feasible without the need for stable isotope labeling techniques.     

Analysis of protein phosphorylation by mass spectrometry

The reversible phosphorylation of proteins is recognized as an essential post-translational modification regulating cell signaling and ultimately function of biological systems. Detection of phosphopeptides and localization of phosphorylation sites remains quite a challenge, even if the protein is purified to near homogeneity. Mass spectrometry has become a vital technique that is routinely utilized for the identification of proteins from whole cell lysates. Nonetheless, due to the minimal amount of phosphorylation found on proteins, enrichment steps for isolating phosphopeptides from complex mixtures have been the focus of many research groups world-wide. In this review, we describe some current methods for the enrichment of phosphopeptides that are compatible with mass spectrometry for assignment of phosphorylation sites. Phosphorylation modifications on proteins and peptides are either directly isolated by solid-phase approaches or chemically modified for selective isolation and/or improved characterization by mass spectrometry. These strategies hold the potential for rapid and sensitive profiling of phosphoproteins from a variety of sources and cellular conditions.     

Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry

Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.     

Site-specific phosphorylation profiling of Arabidopsis proteins by mass spectrometry and peptide chip analysis

An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear-enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins. Among 21 different PKs, 12 were phosphorylated in their activation loops, suggesting that they were in their active state. Immunoblotting and mutational analysis confirmed a tyrosine phosphorylation site in the activation loop of a GSK3/shaggy-like kinase. Analysis of phosphorylation motifs in the substrates suggested links between several of these PKs and many target sites. To perform quantitative phosphorylation analysis, peptide arrays were generated with peptides corresponding to in vivo phosphorylation sites. These peptide chips were used for kinome profiling of subcellular fractions as well as H 2O 2-treated Arabidopsis cells. Different peptide phosphorylation profiles indicated the presence of overlapping but distinct PK activities in cytosolic and nuclear compartments. Among different H 2O 2-induced PK targets, a peptide of the serine/arginine-rich (SR) splicing factor SCL30 was most strongly affected. SRPK4 (SR protein-specific kinase 4) and MAPKs (mitogen-activated PKs) were found to phosphorylate this peptide, as well as full-length SCL30. However, whereas SRPK4 was constitutively active, MAPKs were activated by H 2O 2. These results suggest that SCL30 is targeted by different PKs. Together, our data demonstrate that a combination of mass spectrometry with peptide chip phosphorylation profiling has a great potential to unravel phosphoproteome dynamics and to identify PK substrates.     

Targeted analysis of tyrosine phosphorylation by immuno-affinity enrichment of tyrosine phosphorylated peptides prior to mass spectrometric analysis

Tyrosine phosphorylation is a key process that regulates seminal biological functions, hence, deregulation of this mechanism is an underlying cause of several diseases including cancer and immunological disorders. Due to its low abundance, tyrosine phosphorylation is typically under-represented in most of the global MS-based phosphoproteomic studies. Here, we describe a selective approach based on immuno-affinity purification using specific antibodies to enrich tyrosine phosphorylated peptides from a complex proteolytic digest. LC-MS/MS analysis is subsequently used for peptide identification allowing the exact localization of the phosphorylated residue within the sequence. Using this approach more than 1000 non-redundant phosphotyrosine peptides can be identified in less than 6h of MS analysis, reflecting the high sensitivity and specificity of the technique. The identified tyrosine phosphorylated peptides can be used to study different biological aspects of tyrosine signaling and disease.     

Label-free liquid chromatography–tandem mass spectrometry analysis with automated phosphopeptide enrichment reveals dynamic human milk protein phosphorylation during lactation

Protein phosphorylation is a critical posttranslational modification that affects cell–cell signaling and protein function. However, quantifying the relative site-specific changes of phosphorylation occupancies remains a major issue. An online enrichment of phosphopeptides using titanium dioxide incorporated in a microchip liquid chromatography device was used to analyze trypsin-digested human milk proteins with mass spectrometry. The method was validated with standards and used to determine the dynamic behavior of protein phosphorylation in human milk from the first month of lactation. α-Casein, β-casein, osteopontin, and chordin-like protein 2 phosphoproteins were shown to vary during this lactation time in an independent manner. In addition, changes in specific regions of these phosphoproteins were found to vary independently. Novel phosphorylation sites were discovered for chordin-like protein 2, α-lactalbumin, β-1,4-galactosyl transferase, and poly-Ig (immunoglobulin) receptor. Coefficients of variation for the quantitation were comparable to those in other contemporary approaches using isotopically labeled peptides, with a median value of 11% for all phosphopeptide occupancies quantified.     

Correlation between phosphorylation ratios by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis and radioactivities by radioactive assay

To investigate the correlation between the counts per minute (CPM) by radioactivity assay and the phosphorylation ratio by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, we prepared 136 peptide substrates. The correlation coefficient of phosphorylation ratios to CPM was 0.77 for all samples. However, the more the numbers of positively charged amino acids increased, the more the correlation coefficient increased. Although positively charged amino acids can have an effect on the correlation results, MALDI-TOF MS analysis is a useful means for monitoring phosphorylated peptide and protein kinase activity instead of radioactivity assays.     

Tandem mass spectrometry: a novel approach for metabolic flux analysis

The goal of metabolic flux analysis (MFA) is the accurate estimation of intracellular fluxes in metabolic networks. Here, we introduce a new method for MFA based on tandem mass spectrometry (MS) and stable-isotope tracer experiments. We demonstrate that tandem MS provides more labeling information than can be obtained from traditional full scan MS analysis and allows estimation of fluxes with better precision. We present a modeling framework that takes full advantage of the additional labeling information obtained from tandem MS for MFA. We show that tandem MS data can be computed for any network model, any compound and any tandem MS fragmentation using linear mapping of isotopomers. The inherent advantages of tandem MS were illustrated in two network models using simulated and literature data. Application of tandem MS increased the observability of the models and improved the precision of estimated fluxes by 2- to 5-fold compared to traditional MS analysis.     

The application of hybrid mass spectrometry/mass spectrometry and high-resolution mass spectrometry to the analysis of fish samples for polychlorinated dibenzo-p-dioxins and dibenzofurans

A hybrid mass spectrometer operated in low-resolution selected decomposition monitoring (SDM) mode was used for the analysis of whole fish samples for the 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and poly-chlorinated dibenzofurans (PCDD/Fs). These fish samples were previously analyzed for PCDD/Fs using a high-resolution mass spectrometer following EPA protocols. The hybrid tandem mass spectrometric method using loss of COCl gave similar quantification results to those obtained by high-resolution mass spectrometry and eliminated the interferences attributed to polychlorinated biphenyls that were encountered in the high-resolution mass spectrometric analysis. Comparison of the two methods shows that the high-resolution mass spectrometric method surpasses the tandem mass spectrometric method in other analytical figures of merit, such as detection limit, linearity and reproducibility.     

Plant protein phosphorylation monitored by capillary liquid chromatography--element mass spectrometry

Many essential cellular functions such as growth rate, motility, and metabolic activity are linked to reversible protein phosphorylation, since they are controlled by signaling cascades based mainly on phosphorylation/dephosphorylation events. Quantification of global or site-specific protein phosphorylation is not straightforward with standard proteomic techniques. The coupling of capillary liquid chromatography (microLC) with ICP-MS (inductively coupled plasma-mass spectrometry) is a method which allows a quantitative screening of protein extracts for their phosphorus and sulfur content, and thus provides access to the protein phosphorylation degree. In extension of a recent pilot study, we analyzed protein extracts from the model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii as representatives for multicellular and unicellular green photosynthetically active organisms. The results indicate that the average protein phosphorylation level of the algae C. reinhardtii is higher than that of A. thaliana. Both the average phosphorylation levels were found to be between the extreme values determined so far for prokaryotes (C. glutamicum, lowest levels) and eukaryotes (Mus musculus, highest levels). Tissue samples of A. thaliana representing different stages of plant development showed varying levels of protein phosphorylation indicating a different adjustment of the kinase/phosphatase system. We also utilized the microLC-ICP-MS technology to estimate the efficiency of a novel phosphoprotein enrichment method based on aluminum hydroxide, since the enrichment of phosphorylated species is often an essential step for their molecular characterization.     

Protein phosphorylation analysis by site-specific arginine-mimic labeling in gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Although recent advances in gel electrophoresis and mass spectrometry have greatly facilitated separation, purification, and identification of proteins, significant challenges remain in relation to phosphoprotein analysis. Here we introduce a powerful method for analysis of protein phosphorylation in which phosphorylation sites are labeled with guanidinoethanethiol (GET) by beta-elimination/Michael addition prior to proteolysis and mass spectrometry (MS) analysis. This technique is especially useful in conjunction with gel-based technology in that all of the processes involved, including GET labeling, washing, and phosphospecific enzymatic hydrolysis, can be carried out in excised gel slices, thereby minimizing sample loss and contamination. The novel GET tag, which has a highly basic guanidine group, increases the peak intensities for the GET-labeled tryptic peptides by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. In addition, phosphospecific proteolytic cleavage occurs at guanidinoethylcysteine (Gec) residue, which is arginine-mimic formed by GET tagging of phosphorylated serine residues. Thus, GET tagging is especially useful in analysis of long tryptic phosphopeptides. To illustrate the utility of the in-gel GET tagging and digestion approach, we used it to precisely analyze the phosphorylation sites of human glutathione S-transferase P1 (GSTP1), an enzyme involved in phase II metabolism of many carcinogens and anticancer drugs. The in-gel GET tagging/digestion technique significantly enhances the analytical potential of gel electrophoresis/MS in studies of proteome phosphorylation.     

Immunoaffinity enrichments followed by mass spectrometric detection for studying global protein tyrosine phosphorylation

Phosphorylation of protein tyrosine residues regulates important cell functions and is, when dysregulated, often crucially involved in oncogenesis. It is therefore important to develop and evaluate methods for identifying and studying tyrosine phosphorylated (P-Tyr) proteins. P-Tyr proteins are present at very low concentrations within cells, requiring highly selective enrichment methods to be detected. In this study, we applied immunoaffinity as enrichment step for P-Tyr proteins. Five selected anti-phosphotyrosine antibodies (monoclonal antibodies 4G10, PY100, PYKD1, 13F9 and one polyclonal antiserum) were evaluated with respect to their capability to enrich P-Tyr proteins from cell extracts of the K562 leukemia cell line. The enrichment resulted in the detection of a group of proteins that potentially were tyrosine-phosphorylated (putative P-Tyr proteins). High accuracy identification of actual P-Tyr sites were performed using a highly selective and sensitive liquid chromatography Fourier transform mass spectrometer (LC-FTMS) setup with complementary collision activated dissociation (CAD) and electron capture dissociation (ECD) fragmentations. 4G10 and PY100 antibodies recognized the greatest number of putative P-Tyr proteins in initial screening experiments and were therefore further evaluated and compared in immunoaffinity enrichment of both P-Tyr proteins and peptides. Using the 4G10 antibody for enrichment of proteins, we identified 459 putative P-Tyr proteins by MS. Out of these proteins, 12 were directly verified as P-Tyr proteins by MS analysis of the actual site. Using the PY100 antibody for enrichment of peptides, we detected 67 P-Tyr peptides (sites) and 89 putative P-Tyr proteins. Generally, enrichment at the peptide level made it difficult to reliably determine the identity of the proteins. In contrast, protein identification following immunoaffinity enrichment at the protein level gave greater sequence coverage and thus a higher confidence in the protein identification. By combining all available information, 40 proteins were identified as true P-Tyr proteins from the K562 cell line. In conclusion, this study showed that a combination of immunoaffinity enrichment using multiple antibodies of both intact and digested proteins in parallel experiments is required for best possible coverage of all possible P-Tyr proteins in a sample.     

The decomposition of benzodiazepines during analysis by capillary gas chromatography/mass spectrometry

A capillary gas chromatography column directly interfaced to a mass spectrometer was used for the analysis of sixteen benzodiazepines. The thermal stability of the drugs was found to be related to their chemical structure. Nine of the benzodiazepines were thermally unstable indicating that care should be taken in the interpretation of gas chromatographic data from this class of drugs. The unstable benzodiazepines were: ketazolam which decomposes to diazepam; N-4 oxides (chlordiazepoxide and demoxepam) which lose an oxygen radical; aromatic 7-nitro compounds (nitrazepam and clonazepam) which are partially reduced to the corresponding amine; alpha-hydroxy ketones (lorazepam and oxazepam) which decompose with the loss of water and N-methyl-alpha-hydroxy ketones (lormetazepam and temazepam) which partially decompose with the loss of a hydrogen molecule to produce the corresponding alpha, beta-diketones. Few problems were encountered in distinguishing the drugs by their mass spectra, the exceptions being ketazolam which decomposes to diazepam and demoxepam which decomposes to desmethyldiazepam. In general, good spectra were obtained from 20-50 ng of drug injected. However, for those compounds where the decompositions were not quantitative (nitrazepam, clonazepam, lormetazepam, temazepam) detection limits were poor.     

Systematic analysis of the epidermal growth factor receptor by mass spectrometry reveals stimulation-dependent multisite phosphorylation

Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions. Definition of phosphorylation sites and elucidation of the functional interplay between multiple phosphorylated residues in proteins are, however, a major analytical challenge in current molecular cell biology and proteomic research. In the present study, we used mass spectrometry to determine the major phosphorylated residues of the human epidermal growth factor (EGF) receptor at various well defined cellular conditions. Activation of EGF receptor was achieved by several types of stimulation, i.e. by sodium pervanadate, EGF, and integrin-dependent adhesion. The contribution of cell-matrix adhesion was also determined by activating the EGF receptor by EGF in cells kept in suspension. We developed an analytical strategy that combined miniaturized sample preparation techniques and MALDI tandem mass spectrometry and determined a total of nine phosphorylation sites in the EGF receptor. We discovered one novel phosphorylation site (Ser967) and revealed constitutive phosphorylation of Thr669, Ser967, Ser1002, and Tyr1045 and stimulation-dependent differential phosphorylation of Tyr1068, Tyr1086, Ser1142, Tyr1148, and Tyr1173. The EGF receptor was purified from HeLa cells or ECV304 cells by immunoprecipitation and SDS-PAGE and then digested with trypsin. Phosphopeptides in the range of 0.8-3.7 kDa were recovered by combinations of IMAC, perfusion chromatography, and graphite powder chromatography and subsequently detected and sequenced by MALDI quadrupole time-of-flight tandem mass spectrometry. Two phosphorylation sites were detected in the peptide 1137GSHQISLDNPDYQQDFFPK1155; however, only Tyr1148 was phosphorylated upon EGF treatment; in contrast Ser1142 was only phosphorylated by integrin-dependent adhesion in the absence of EGF treatment, suggesting differential phosphorylation of this region by distinct stimuli. This MALDI MS/MS-based analytical approach demonstrates the feasibility of systematic analysis of signaling molecules by mass spectrometry and provides new insights into the dynamics of receptor signaling processes.     

糖链的生物质谱分析

糖链是重要的生物信息分子,在许多生理和病理过程中都发挥着独特作用。糖链结构非常复杂,具有微观不均一性,其分析和结构解析一直是糖生物学研究的瓶颈。质谱具有灵敏度高、可获得多种结构信息和适于分析混合物等优点,是糖链定性定量分析的一种理想手段。电喷雾电离质谱和基质辅助激光解析电离质谱两大生物质谱技术已被广泛应用于糖链的相对分子质量指纹谱分析、序列和连接方式测定及相对定量分析。对近年来以质谱为主要分析手段的糖链分析方法研究进展做一综述。     

swissPIT: a novel approach for pipelined analysis of mass spectrometry data

The identification and characterization of peptides from tandem mass spectrometry (MS/MS) data represents a critical aspect of proteomics. Today, tandem MS analysis is often performed by only using a single identification program achieving identification rates between 10-50% (Elias and Gygi, 2007). Beside the development of new analysis tools, recent publications describe also the pipelining of different search programs to increase the identification rate (Hartler et al., 2007; Keller et al., 2005). The Swiss Protein Identification Toolbox (swissPIT) follows this approach, but goes a step further by providing the user an expandable multi-tool platform capable of executing workflows to analyze tandem MS-based data. One of the major problems in proteomics is the absent of standardized workflows to analyze the produced data. This includes the pre-processing part as well as the final identification of peptides and proteins. The main idea of swissPIT is not only the usage of different identification tool in parallel, but also the meaningful concatenation of different identification strategies at the same time. The swissPIT is open source software but we also provide a user-friendly web platform, which demonstrates the capabilities of our software and which is available at http://swisspit.cscs.ch upon request for account.