Characterization of human myotubes from type 2 diabetic and nondiabetic subjects using complementary quantitative mass spectrometric methods.

Skeletal muscle is a key tissue site of insulin resistance in type 2 diabetes. Human myotubes are primary skeletal muscle cells displaying both morphological and biochemical characteristics of mature skeletal muscle and the diabetic phenotype is conserved in myotubes derived from subjects with type 2 diabetes. Several abnormalities have been identified in skeletal muscle from type 2 diabetic subjects, however, the exact molecular mechanisms leading to the diabetic phenotype has still not been found. Here we present a large-scale study in which we combine a quantitative proteomic discovery strategy using isobaric peptide tags for relative and absolute quantification (iTRAQ) and a label-free study with a targeted quantitative proteomic approach using selected reaction monitoring to identify, quantify, and validate changes in protein abundance among human myotubes obtained from nondiabetic lean, nondiabetic obese, and type 2 diabetic subjects, respectively. Using an optimized protein precipitation protocol, a total of 2832 unique proteins were identified and quantified using the iTRAQ strategy. Despite a clear diabetic phenotype in diabetic myotubes, the majority of the proteins identified in this study did not exhibit significant abundance changes across the patient groups. Proteins from all major pathways known to be important in type 2 diabetic subjects were well-characterized in this study. This included pathways like the trichloroacetic acid (TCA) cycle, lipid oxidation, oxidative phosphorylation, the glycolytic pathway, and glycogen metabolism from which all but two enzymes were found in the present study. None of these enzymes were found to be regulated at the level of protein expression or degradation supporting the hypothesis that these pathways are regulated at the level of post-translational modification. Twelve proteins were, however, differentially expressed among the three different groups. Thirty-six proteins were chosen for further analysis and validation using selected reaction monitoring based on the regulation identified in the iTRAQ discovery study. The abundance of adenosine deaminase was considerably down-regulated in diabetic myotubes and as the protein binds propyl dipeptidase (DPP-IV), we speculate whether the reduced binding of adenosine deaminase to DPP-IV may contribute to the diabetic phenotype in vivo by leading to a higher level of free DPP-IV to bind and inactivate the anti-diabetic hormones, glucagon-like peptide-1 and glucose-dependent insulintropic polypeptide.

Technologies for plasma membrane proteomics.

Cell-cell and intracellular signaling are critical mechanisms by which an organism can respond quickly and appropriately to internal or environmental stimuli. Transmission of the stimulus to effector proteins must be coordinated, rapid and transient such that the response is not exaggerated and the overall balance of the cell or tissue is retained. Proteomics technology has traditionally been adept at analyzing effector proteins (such as cytoskeletal and heat shock proteins, and those involved in metabolic processes) in studies examining the effects of altered environmental or nutritional conditions, drugs, or genetic manipulation, since these proteins are often highly abundant, soluble and therefore amenable to analysis. Conversely, the proteins mediating the transmission of the signal have been generally under-represented, typically because of their low abundance. One mechanism that has overcome this to some extent is the advent of very high-resolution phosphoproteomics techniques, which have enabled temporal profiling of intracellular signal pathways via quantitative assessment of peptide phosphorylation sites. One group of proteins, however, that still remains under-represented in proteomics studies are those found in the plasma membrane (PM). Such proteins are crucial in sensing changes in the external environment and in stimulating the transmission of the signal intracellularly. This review examines PM proteins and appraises the proteomics approaches currently available for providing a comprehensive analysis of these crucial mediators of signal pathways. We discuss different strategies for enrichment and solubilization of these proteins and include discussion on cross-linking of PM complexes and glycoproteomics as the basis for purification prior to proteomic analyses.

Undesirable charge-enhancement of isobaric tagged phosphopeptides leads to reduced identification efficiency.

The study of cellular dynamics by proteomics using mass spectrometry requires a quantitation strategy that is robust, sensitive, and of sufficient resolution to deal with subtle changes in protein expression or post-translational modification. The major quantitation strategies are stable isotopic labeling of proteins and peptides for in vitro cell culture systems (stable isotope labeling using amino acids in cell culture, SILAC) or isobaric peptide labels such as isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMT) for both in vitro and in vivo systems. These quantitation strategies have also been successfully applied to phosphoproteomics studies for the investigation of signal transduction pathways. Here we describe major drawbacks associated with isobaric labeling for the identification and quantitation of phosphopeptides using electrospray tandem mass spectrometry. Phosphopeptide derivatization with isobaric tags results in significantly greater charging in electrospray ionization. This reduces phosphopeptide identification efficiency with multistage activation and HCD MS/MS by more than 50% and may contribute to the discrepancy observed between identifications observed for large cell- or tissue-based data sets from labeled and nonlabeled peptide mixtures. Ammonia vapor sprayed perpendicular to the electrospray needle during ionization resulted in an overall decrease in the average charge states and a concomitant increase in phosphopeptide identifications.

Strategies for quantitation of phosphoproteomic data.

Recent developments in phosphoproteomic sample-preparation techniques and sensitive mass spectrometry instrumentation have led to large-scale identifications of phosphoproteins and phosphorylation sites from highly complex samples. This has facilitated the implementation of different quantitation strategies in order to study the biological role of protein phosphorylation during disease progression, differentiation or during external stimulation of a cellular system. In this article, a brief summary of the most popular strategies for phosphoproteomic studies is given; however, the main focus will be on different quantitation strategies. Methods for metabolic labeling, chemical modification and label-free quantitation and their applicability or inapplicability in phosphoproteomic studies are discussed.

Quantitative iTRAQ-Based Proteomic Identification of Candidate Biomarkers for Diabetic Nephropathy in Plasma of Type 1 Diabetic Patients.

INTRODUCTION: As part of a clinical proteomics programme focused on diabetes and its complications, it was our goal to investigate the proteome of plasma in order to find improved candidate biomarkers to predict diabetic nephropathy. METHODS: Proteins derived from plasma from a cross-sectional cohort of 123 type 1 diabetic patients previously diagnosed as normoalbuminuric, microalbuminuric or macroalbuminuric were enriched with hexapeptide library beads and subsequently pooled within three groups. Proteins from the three groups were compared by online liquid chromatography and tandem mass spectrometry in three identical repetitions using isobaric mass tags (iTRAQ). The results were further analysed with ingenuity pathway analysis. Levels of apolipoprotein A1, A2, B, C3, E and J were analysed and validated by a multiplex immunoassay in 20 type 1 diabetic patients with macroalbuminuria and 10 with normoalbuminuria. RESULTS: A total of 112 proteins were identified in at least two out of three replicates. The global protein ratios were further evaluated by ingenuity pathway analysis, resulting in the recognition of apolipoprotein A2, B, C3, D and E as key nodes in the top-rated network. The multiplex immunoassay confirmed the overall protein expression patterns observed by the iTRAQ analysis. CONCLUSION: The candidate biomarkers discovered in this cross-sectional cohort may turn out to be progression biomarkers and might have several clinical applications in the treatment and monitoring of diabetic nephropathy; however, they need to be confirmed in a longitudinal cohort. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12014-010-9053-0) contains supplementary material, which is available to authorized users.

SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides.

The complete analysis of phosphoproteomes has been hampered by the lack of methods for efficient purification, detection, and characterization of phosphorylated peptides from complex biological samples. Despite several strategies for affinity enrichment of phosphorylated peptides prior to mass spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide, the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy, SIMAC (sequential elution from IMAC), for sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This allows individual analysis of the two pools of phosphorylated peptides using mass spectrometric parameters differentially optimized for their unique properties. We compared the phosphoproteome identified from 120 mug of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a 3-fold increase in recovery of multiply phosphorylated peptides.

Loss of mismatch repair signaling impairs the WNT-bone morphogenetic protein crosstalk and the colonic homeostasis.

The fine balance between proliferation, differentiation, and apoptosis in the colonic epithelium is tightly controlled by the interplay between WNT, Notch, and bone morphogenetic protein (BMP) signaling. How these complex networks coordinate the colonic homeostasis, especially if cancer predisposing mutations such as mutations in the DNA mismatch repair (MMR) are present, is unclear. Inactivation of the MMR system has long been linked to colorectal cancer; however, little is known about its role in the regulation of the colonic homeostasis. It has been shown that loss of MMR promotes the proliferation of colon epithelial cells that renders them highly susceptible to transformation. The mechanism through which MMR mediates this effect, yet, remains to be determined. Using an MMR-deficient mouse model, we show that increased methylation of Dickkopf1 impacts its expression, and consequently, the ability to negatively regulate WNT signaling. As a result, excessive levels of active ß-catenin promote strong crypt progenitor-like phenotype and abnormal proliferation. Under these settings, the development and function of the goblet cells are affected. MMR-deficient mice have fewer goblet cells with enlarged mucin-loaded vesicles. We further show that MMR inactivation impacts the WNT-BMP signaling crosstalk.

Analytical strategies for phosphoproteomics.

Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phospho-specific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.

Enrichment and characterization of phosphopeptides by immobilized metal affinity chromatography (IMAC) and mass spectrometry.

The combination of immobilized metal affinity chromatography (IMAC) and mass spectrometry is a widely used technique for enrichment and sequencing of phosphopeptides. In the IMAC method, negatively charged phosphate groups interact with positively charged metal ions (Fe3+, Ga3+, and Al3+) and this interaction makes it possible to enrich phosphorylated peptides from rather complex peptide samples. Phosphopeptide enrichment by IMAC is sensitive and specific for peptide mixtures derived from pure proteins or simple protein mixtures. The selectivity of the IMAC method is, however, limited when working with peptide mixtures derived from highly complex samples, e.g., whole-cell extracts, where sample prefractionation is advisable. Furthermore, lowering the pH value of the sample loading buffer reduces nonspecific binding to the IMAC resin significantly, thereby improving the selectivity of IMAC for phosphopeptides. The retained phosphopeptides are released from the IMAC resin by using alkaline buffers (pH 10-11), EDTA, or phosphate-containing buffers. We have described a detailed and robust protocol for IMAC for phosphopeptide enrichment from semi-complex mixtures.

The use of titanium dioxide micro-columns to selectively isolate phosphopeptides from proteolytic digests.

Titanium dioxide has very high affinity for phosphopeptides and it has become an efficient alternative to already existing methods for phosphopeptide enrichment from complex samples. Peptide loading in a highly acidic environment in the presence of 2,5-dihydroxybenzoic acid (DHB), phthalic acid, or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding from nonphosphorylated peptides. The enriched phosphopeptides bound to the titanium dioxide are subsequently eluted from the micro-column using an alkaline buffer. Titanium dioxide chromatography is extremely tolerant towards most buffers used in biological experiments. It is highly robust and as such it has become one of the methods of choice in large-scale phospho-proteomics. Here we describe the protocol for phosphopeptide enrichment using titanium dioxide chromatography followed by desalting and concentration of the sample by reversed phase chromatography prior to MS analysis.

Enrichment and separation of mono- and multiply phosphorylated peptides using sequential elution from IMAC prior to mass spectrometric analysis.

Phospho-proteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., immobilized metal affinity chromatography and titanium dioxide chromatography, provide varying degrees of selectivity and specificity for phosphopeptide enrichment. Furthermore, the number of multiply phosphorylated peptides that are identified in most published studies is rather low. Here the protocol for a new strategy that separates mono-phosphorylated pep-tides from multiply phosphorylated peptides using sequential elution from immobilized metal affinity chromatography is described. The two separate phosphopeptide fractions are subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multiply phosphorylated peptides, respectively, resulting in improved identification of especially multiply phosphorylated peptides from a minimum amount of starting material. The new method increases the coverage of the phosphoproteome significantly.

Analysis of posttranslational modifications of proteins by tandem mass spectrometry.

Protein activity and turnover is tightly and dynamically regulated in living cells. Whereas the three-dimensional protein structure is predominantly determined by the amino acid sequence, posttranslational modification (PTM) of proteins modulates their molecular function and the spatial-temporal distribution in cells and tissues. Most PTMs can be detected by protein and peptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful for the characterization of modified proteins via amino acid sequencing and specific detection of posttranslationally modified amino acid residues. Large-scale, quantitative analysis of proteins by MS/MS is beginning to reveal novel patterns and functions of PTMs in cellular signaling networks and biomolecular structures.

Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.

The Use of Titanium Dioxide for Selective Enrichment of Phosphorylated Peptides.

Titanium dioxide (TiO2) has very high affinity for phosphopeptides and in recent years it has become one of the most popular methods for phosphopeptide enrichment from complex biological samples. Peptide loading onto TiO2 resin in a highly acidic environment in the presence of 2,5-dihydroxybenzoic acid (DHB), phthalic acid, lactic acid, or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding of non-phosphorylated peptides. The phosphopeptides bound to the TiO2 are subsequently eluted from the chromatographic material using an alkaline buffer. TiO2 chromatography is extremely tolerant towards most buffers used in biological experiments, highly robust and as such it has become the method of choice in large-scale phosphoproteomics. Here we describe a batch mode protocol for phosphopeptide enrichment using TiO2 chromatographic material followed by desalting and concentration of the sample by reversed phase micro-columns prior to downstream MS and LC-MS/MS analysis.

Sequential Elution from IMAC (SIMAC): An Efficient Method for Enrichment and Separation of Mono- and Multi-phosphorylated Peptides.

Phosphoproteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems, especially when using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., Immobilized Metal ion Affinity Chromatography and titanium dioxide chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. The number of multi-phosphorylated peptides identified in most published studies is rather low. Here we describe a protocol for a strategy that separates mono-phosphorylated peptides from multiply phosphorylated peptides using Sequential elution from Immobilized Metal ion Affinity Chromatography. The method relies on the initial enrichment and separation of mono- and multi-phosphorylated peptides using Immobilized Metal ion Affinity Chromatography and a subsequent enrichment of the mono-phosphorylated peptides using titanium dioxide chromatography. The two separate phosphopeptide fractions are then subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multi-phosphorylated peptides, respectively, resulting in improved identification of especially multi-phosphorylated peptides from a minimum amount of starting material.

TiO(2)-based phosphoproteomic analysis of the plasma membrane and the effects of phosphatase inhibitor treatment.

Phosphorylation of plasma membrane proteins frequently initiates signal transduction pathways or attenuate plasma membrane transport processes. Because of the low abundance and hydrophobic features of many plasma membrane proteins and the low stoichiometry of protein phosphorylation, studies of the plasma membrane phosphoproteome are challenging. We present an optimized analytical strategy for plasma membrane phosphoproteomics that combines efficient plasma membrane protein preparation with TiO(2)-based phosphopeptide enrichment and high-performance mass spectrometry for phosphopeptide sequencing. We used sucrose centrifugation in combination with sodium carbonate extraction to achieve efficient and reproducible purification of low microgram levels of plasma membrane proteins from human mesenchymal stem cells (hMSCs, 10(7) cells), achieving more than 70% yield of membrane proteins. Phosphopeptide enrichment by titanium dioxide chromatography followed by capillary liquid chromatography-tandem mass spectrometry allowed us to assign 703 unique phosphorylation sites in 376 phosphoproteins. Our experiments revealed that treatment of cell cultures with three different types of protein phosphatase inhibitors produces distinct phosphopeptide populations and an increase of 10-40% of the number of detected and sequenced phosphoserine, phosphothreonine and phosphotyrosine containing peptides. In summary, our analytical strategy enables functional phosphoproteomic analysis of stem cell differentiation and cell surface biomarker discovery using very low amounts of starting material.

Highly selective enrichment of phosphorylated peptides using titanium dioxide.

The characterization of phosphorylated proteins is a challenging analytical task since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric. Highly efficient enrichment procedures are therefore required. Here we describe a protocol for selective phosphopeptide enrichment using titanium dioxide (TiO2) chromatography. The selectivity toward phosphopeptides is obtained by loading the sample in a 2,5-dihydroxybenzoic acid (DHB) or phthalic acid solution containing acetonitrile and trifluoroacetic acid (TFA) onto a TiO2 micro-column. Although phosphopeptide enrichment can be achieved by using TFA and acetonitrile alone, the selectivity is dramatically enhanced by adding DHB or phthalic acid since these compounds, in conjunction with the low pH caused by TFA, prevent binding of nonphosphorylated peptides to TiO2. Using an alkaline solution (pH > or = 10.5) both monophosphorylated and multiphosphorylated peptides are eluted from the TiO2 beads. This highly efficient method for purification of phosphopeptides is well suited for the characterization of phosphoproteins from both in vitro and in vivo studies in combination with mass spectrometry (MS). It is a very easy and fast method. The entire protocol requires less than 15 min per sample if the buffers have been prepared in advance (not including lyophilization).

Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.

Reversible phosphorylation of proteins regulates the majority of all cellular processes, e.g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation is often substoichiometric, and an enrichment procedure of phosphorylated peptides derived from phosphorylated proteins is a necessary prerequisite for the characterization of such peptides by modern mass spectrometric methods. We report a highly selective enrichment procedure for phosphorylated peptides based on TiO2microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO2. We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented.