Tyrosine phosphorylation profiling in FGF-2 stimulated human embryonic stem cells

10.1371/journal.pone.0017538PLoS ONE63

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

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

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

Measuring protein structural changes on a proteome-wide scale using limited proteolysis-coupled mass spectrometry

Protein structural changes induced by external perturbations or internal cues can profoundly influence protein activity and thus modulate cellular physiology. A number of biophysical approaches are available to probe protein structural changes, but these are not applicable to a whole proteome in a biological extract. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a recently developed proteomics approach that enables the identification of protein structural changes directly in their complex biological context on a proteome-wide scale. After perturbations of interest, proteome extracts are subjected to a double-protease digestion step with a nonspecific protease applied under native conditions, followed by complete digestion with the sequence-specific protease trypsin under denaturing conditions. This sequential treatment generates structure-specific peptides amenable to bottom-up MS analysis. Next, a proteomics workflow involving shotgun or targeted MS and label-free quantification is applied to measure structure-dependent proteolytic patterns directly in the proteome extract. Possible applications of LiP-MS include discovery of perturbation-induced protein structural alterations, identification of drug targets, detection of disease-associated protein structural states, and analysis of protein aggregates directly in biological samples. The approach also enables identification of the specific protein regions involved in the structural transition or affected by the binding event. Sample preparation takes approximately 2 d, followed by one to several days of MS and data analysis time, depending on the number of samples analyzed. Scientists with basic biochemistry training can implement the sample preparation steps. MS measurement and data analysis require a background in proteomics

Supplementary Material for: Emergency Laparoscopic Sigmoidectomy for Perforated Diverticulitis with Generalised Peritonitis: A Systematic Review

<br><strong><em>Background:</em></strong> Laparoscopic sigmoidectomy for diverticulitis has initially been confined to the elective setting. However, open acute sigmoidectomy for perforated diverticulitis is associated with high morbidity rates that might be reduced after laparoscopic surgery. The aim of this systematic review was to assess the feasibility of emergency laparoscopic sigmoidectomy for perforated diverticulitis. <b><i>Methods:</i></b> We performed a systematic search of PubMed, EMBASE and CENTRAL. All studies reporting on patients with perforated diverticulitis (Hinchey III-IV) treated by laparoscopic sigmoidectomy in the acute phase were included, regardless of design. <b><i>Results:</i></b> We included 4 case series and one cohort study (total of 104 patients) out of 1,706 references. Hartmann's procedure (HP) was performed in 84 patients and primary anastomosis in 20. The mean operating time varied between 115 and 200 min. The conversion rate varied from 0 to 19%. The mean length of hospital stay ranged between 6 and 16 days. Surgical re-intervention was necessary in 2 patients. In 20 patients operated upon without defunctioning ileostomy, no anastomotic leakage was reported. Three patients died during the postoperative period. Stoma reversal after HP was performed in 60 out of 79 evaluable patients (76%). <b><i>Conclusions:</i></b> Acute laparoscopic sigmoidectomy for the treatment of perforated diverticulitis is feasible in selected patients provided they are handled by experienced hands