Advancing liquid chromatography- mass spectrometry based technologies for proteome research

In proteomics, high-tech nano-liquid chromatography (LC) and mass spectrometry (MS) instrumentation is used to routinely sequence proteins at a large scale. In this thesis, several technological developments are described to advance proteomics and their applicability is demonstrated in several different research lines. HILIC is an LC phase that exhibits some features that can be utilized in proteomics. The orthogonality of separation with reversed phase-LC and the more even dispersion of peptides over the LC run, makes HILIC an adequate first dimension for the fractionation of complex peptide mixtures. A specific variety of HILIC has been evaluated and further optimized for two dimensional-LC; zwitterionic HILIC (ZIC-HILIC). A mixed mode separation was observed for ZIC-HILIC consisting of both electrostatic and polar interactions between the peptides and stationary phase. Compared to SCX, less clustering of the typically ubiquitous +2 and +3 charged peptides was observed. Furthermore, the development of a triplex stable isotope dimethyl labeling approach is reported. By using different isotopomers of formaldehyde and cyanoborohydride, three different dimethyl labels can be generated in order to simultaneously analyze peptides from three different samples by LC-MS. The approach uses cheap and readily available chemicals and therefore large amounts of sample can be labeled. Furthermore, the labeling reaction goes to close to completion and virtually any sample can be labeled as the sample is performed post-lysis and -digestion. The metalloendopeptidase Lys-N has been investigated for MALDI-MS/MS proteomics applications. Lys-N produces peptides with an N-terminal Lys residue and therefore the basicity is concentrated at the N-terminus. Fragmentation in MALDI of peptides where this N-terminal Lys is the only basic group results in the generation of primarily N-terminal fragments. The CID spectra are straightforward and the sequence can be easily read off since often complete sequence ladders of b-ions are present. Phosphorylated peptides typically fragment differently compared to non-modified peptides. In CID for example, fragmentation of phosphorylated peptides can result in spectra that are dominated by a single peak that represents the neutral loss of the phosphate group. In chapter 6, this neutral loss effect and ways to accommodate challenges of MS based analysis of phosphopeptides are reviewed. The analysis of Tyr phosphorylation is rather challenging due to the low levels of Tyr phosphorylation. The enrichment efficiency of a phosphopeptide immuno-affinity purification was shown to be rather high and more than 1000 phospho-Tyr peptides could be identified after two separate LC-MS runs. Triplex stable isotope dimethyl labeling was combined with phospho-Tyr immunoprecipitation. Tyr phosphorylation was profiled after pervanadate and EGF stimulation, respectively. The quantitative immunoprecipitation method was also applied to study the role of FGF-2 stimulation in human embryonic stem cells (hESCs). FGF-2 is important in the maintenance of the pluripotency state of hESCs. Several hundreds of Tyr phosphorylation sites could be identified and quantified of which some 140 showed a differential regulation upon FGF-2 stimulation. These regulated sites were found on proteins that include FGF receptors, members of the Src family, proteins involved in actin polymerization and cyclin dependent kinases

Cohabitation, an alternative to marriage ? A cross-national study 1983

Comparing heterosexual cohabitating couples with corresponding married couples ( 'matching' ) between 20 and 40 years old, on some social and psychological characteristics, in the Netherlands and in the USA. Duration of relationship / separate or joint responsibility for lodging, finances / approval by others of this kind of relationship / having made formal legal provisions in cases of separation or death / planning children / reasons for living together / reasons for eventual marriage / circumstances under which partner could be left / expectations before starting to live together / statements regarding a variety of issues / relation to parents. Background variables: basic characteristics/ household characteristics/ characteristics of parental family/household/ occupation/employment/ income/capital assets/ education/ religio

Cohabitation, an alternative to marriage ? 1983

Comparing heterosexual cohabitating couples with corresponding married couples ( 'matching' ) between 20 and 40 years old, on some social and psychological characteristics, in the Netherlands and in the USA. Duration of relationship / separate or joint responsibility for lodging, finances / approval by others of this kind of relationship / having made formal legal provisions in cases of separation or death / planning children / reasons for living together / reasons for eventual marriage / circumstances under which partner could be left / expectations before starting to live together / statements regarding a variety of issues / relation to parents. Background variables: basic characteristics/ household characteristics/ characteristics of parental family/household/ occupation/employment/ income/capital assets/ education/ religio

Unambiguous Phosphosite Localization using Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)

We recently introduced a novel scheme combining electron-transfer and higher-energy collision dissociation (termed EThcD), for improved peptide ion fragmentation and identification. We reasoned that phosphosite localization, one of the major hurdles in high-throughput phosphoproteomics, could also highly benefit from the generation of such EThcD spectra. Here, we systematically assessed the impact on phosphosite localization utilizing EThcD in comparison to methods employing either ETD or HCD, respectively, using a defined synthetic phosphopeptide mixture and also using a larger data set of Ti4+-IMAC enriched phosphopeptides from a tryptic human cell line digest. In combination with a modified version of phosphoRS, we observed that in the majority of cases EThcD generated richer and more confidently identified spectra, resulting in superior phosphosite localization scores. Our data demonstrates the distinctive potential of EThcD for PTM localization, also beyond protein phosphorylation

Evaluation and Optimization of ZIC-HILIC-RP as an alternative MudPIT Strategy

In proteomics, a digested cell lysate is often too complex for direct comprehensive mass spectrometric analysis. To reduce complexity, several peptide separation techniques have been introduced including very successful two-dimensional liquid chromatography (2D-LC) approaches. Here, we assess the potential of zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) as a first dimension for the analysis of complex peptide mixtures. We show that ZIC-HILIC separation is dramatically dependent on buffer pH in the range from 3 to 8, due to deprotonation of acidic amino acids. ZIC-HILIC exhibits a mixed-mode effect consisting of electrostatic and polar interactions. We developed a 2D-LC system that hyphenates ZIC-HILIC off-line with reversed-phase (RP). The two dimensions are fairly orthogonal, and the system performs very well in the analysis of minute amounts of complex peptide mixtures. Applying this method to the analysis of 10 mug of a cellular nuclear lysate, we were able to confidently identify over 1000 proteins. Compared to strong cation exchange chromatography (SCX), ZIC-HILIC shows better chromatographic resolution and absence of clustering of prevalent +2 and +3 charged peptides. At pH 3, ZIC-HILIC separation allows best orthogonality with RP and resembles conventional SCX separation. A significant enrichment of N-acetylated peptides in the first fractions is observed at these conditions. ZIC-HILIC separation at high pH (6.8 and 8), however, enables better chromatography, resulting in more comprehensive data acquisition. With this extended flexibility, we conclude that ZIC-HILIC is a very good alternative for the more conventional SCX in multidimensional peptide separation strategies

Quantitative proteomic identification of host factors involved in the Salmonella syphimuruim infectin cycle.

To identify host factors involved in Salmonella replication, SILAC-based quantitative proteomics was used to investigate the interactions of Salmonella typhimurium with the secretory pathway in human epithelial cells. Protein profiles of Golgi-enriched fractions isolated from S. typhimurium-infected cells were compared with those of mock-infected cells, revealing significant depletion or enrichment of 105 proteins. Proteins annotated to play a role in membrane traffic were overrepresented among the depleted proteins whereas proteins annotated to the cytoskeleton showed a diverse behavior with some proteins being enriched, others being depleted from the Golgi fraction upon Salmonella infection. To study the functional relevance of identified proteins in the Salmonella infection cycle, small interfering RNA (siRNA) experiments were performed. siRNA-mediated depletion of a selection of affected proteins identified five host factors involved in Salmonella infection. Depletion of peroxiredoxin-6 (PRDX6), isoform β-4c of integrin β-4 (ITGB4), isoform 1 of protein lap2 (erbin interacting protein; ERBB2IP), stomatin (STOM) or TBC domain containing protein 10b (TBC1D10B) resulted in increased Salmonella replication. Surprisingly, in addition to the effect on Salmonella replication, depletion of STOM or ITGB4 resulted in a dispersal of intracellular Salmonella microcolonies. It can be concluded that by using SILAC-based quantitative proteomics we were able to identify novel host cell proteins involved in the complex interplay between Salmonella and epithelial cells

Toward a comprehensive characterization of a human cancer cell phosphoproteome

Mass spectrometry (MS)-based phosphoproteomics has achieved extraordinary success in qualitative and quantitative analysis of cellular protein phosphorylation. Considering that an estimated level of phosphorylation in a cell is placed at well above 100,000 sites, there is still much room for improvement. Here, we attempt to extend the depth of phosphoproteome coverage while maintaining realistic aspirations in terms of available material, robustness, and instrument running time. We developed three strategies, where each provided a different balance between these three key parameters. The first strategy simply used enrichment by Ti(4+)-IMAC followed by reversed chromatography LC-MS (termed 1D). The second strategy incorporated an additional fractionation step through the use of HILIC (2D). Finally, a third strategy was designed employing first an SCX fractionation, followed by Ti(4+)-IMAC enrichment and additional fractionation by HILIC (3D). A preliminary evaluation was performed on the HeLa cell line. Detecting 3700 phosphopeptides in about 2 h, the 1D strategy was found to be the most sensitive but limited in comprehensivity, mainly due to issues with complexity and dynamic range. Overall, the best balance was achieved using the 2D based strategy, identifying close to 17,000 phosphopeptides with less than 1 mg of material in about 48 h. Subsequently, we confirmed the findings with the K562 cell sample. When sufficient material was available, the 3D strategy increased phosphoproteome allowing over 22,000 unique phosphopeptides to be identified. Unfortunately, the 3D strategy required more time and over 1 mg of material before it started to outperform 2D. Ultimately, combining all strategies, we were able to identify over 16,000 and nearly 24,000 unique phosphorylation sites from the cancer cell lines HeLa and K562, respectively. In summary, we demonstrate the need to carry out extensive fractionation for deep mining of the phosphoproteome and provide a guide for appropriate strategies depending on sample amount and/or analysis time

Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography

Mass spectrometry (MS)-based proteomics has become the preferred tool for the analysis of protein phosphorylation. To be successful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. This is necessary because of the substoichiometric nature of phosphorylation at a given site and the complexity of the cell. Recently, new alternative materials have emerged that allow excellent and robust enrichment of phosphopeptides. These monodisperse microsphere-based immobilized metal ion affinity chromatography (IMAC) resins incorporate a flexible linker terminated with phosphonate groups that chelate either zirconium or titanium ions. The chelated zirconium or titanium ions bind specifically to phosphopeptides, with an affinity that is similar to that of other widely used metal oxide affinity chromatography materials (typically TiO2). Here we present a detailed protocol for the preparation of monodisperse microsphere-based Ti4+-IMAC adsorbents and the subsequent enrichment process. Furthermore, we discuss general pitfalls and crucial steps in the preparation of phosphoproteomics samples before enrichment and, just as importantly, in the subsequent mass spectrometric analysis. Key points such as lysis, preparation of the chromatographic system for analysis and the most appropriate methods for sequencing phosphopeptides are discussed. Bioinformatics analysis specifically relating to site localization is also addressed. Finally, we demonstrate how the protocols provided are appropriate for both single-protein analysis and the screening of entire phosphoproteomes. It takes similar to 2 weeks to complete the protocol: 1 week to prepare the Ti4+-IMAC material, 2 d for sample preparation, 3 d for MS analysis of the enriched sample and 2 d for data analysis