All charged up: High and prolonged yields of multiply charged peptide and protein ions can be formed in MALDI mass spectrometry using liquid UV‐MALDI matrices and a heated ion‐transfer tube. The key ...features are low laser energies of 1–10 μJ, resulting in fluences of less than 200 to 2000 J m−2 and low sample ablation, high sensitivity, and continuous ion generation over tens of thousands of laser shots.
Diabetes like many diseases and biological processes is not mono-causal. On the one hand multi-factorial studies with complex experimental design are required for its comprehensive analysis. On the ...other hand, the data from these studies often include a substantial amount of redundancy such as proteins that are typically represented by a multitude of peptides. Coping simultaneously with both complexities (experimental and technological) makes data analysis a challenge for Bioinformatics.
We present a comprehensive work-flow tailored for analyzing complex data including data from multi-factorial studies. The developed approach aims at revealing effects caused by a distinct combination of experimental factors, in our case genotype and diet. Applying the developed work-flow to the analysis of an established polygenic mouse model for diet-induced type 2 diabetes, we found peptides with significant fold changes exclusively for the combination of a particular strain and diet. Exploitation of redundancy enables the visualization of peptide correlation and provides a natural way of feature selection for classification and prediction. Classification based on the features selected using our approach performs similar to classifications based on more complex feature selection methods.
The combination of ANOVA and redundancy exploitation allows for identification of biomarker candidates in multi-dimensional MALDI-TOF MS profiling studies with complex experimental design. With respect to feature selection our method provides a fast and intuitive alternative to global optimization strategies with comparable performance. The method is implemented in R and the scripts are available by contacting the corresponding author.
Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and ...functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free‐flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) at the protein level or by cation‐exchange high‐performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on‐line liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.∁
Phosphoinositide lipids regulate numerous cellular processes in all eukaryotes. The versatility of this phospholipid is provided by combinations of phosphorylation on the 3′, 4′, and 5′ positions of ...the inositol head group. Two distinct structural families of phosphoinositide (PI) kinases have so far been identified and named after their prototypic members, the PI 3-kinase and phosphatidylinositol (PtdIns) phosphate kinase families, both of which have been found to contain structural homologues possessing PI 4-kinase activity. Nevertheless, the prevalent PtdIns 4-kinase activity in many mammalian cell types is conferred by the widespread type II PtdIns 4-kinase, which has so far resisted molecular characterization. We have partially purified the human type II isoform from plasma membrane rafts of human A431 epidermoid carcinoma cells and obtained peptide mass and sequence data. The results allowed the cDNA containing the full open reading frame to be cloned. The predicted amino acid sequence revealed that the type II enzyme is the prototypic member of a novel, third family of PI kinases. We have named the purified protein type IIα and a second human isoform, type IIβ. The type IIα mRNA appears to be expressed ubiquitously in human tissues, and homologues appear to be expressed in all eukaryotes.
•New AP ionization interface design and its optimization for liquid MALDI MS is described.•Data suggest two distinct ion formation processes in liquid AP-MALDI MS.•Desolvation conditions in AP-MALDI ...ion sources and transfer regions influence the ion signal.•Optimal desolvation conditions are different to those typically applied to nano-ESI plumes.•Optimal ion transfer conditions increased the multiply protonated analyte yield by a factor of >14.
Liquid matrix-assisted laser desorption/ionization (MALDI) allows the generation of predominantly multiply charged ions in atmospheric pressure (AP) MALDI ion sources for mass spectrometry (MS) analysis. The charge state distribution of the generated ions and the efficiency of the ion source in generating such ions crucially depend on the desolvation regime of the MALDI plume after desorption in the AP-to-vacuum inlet. Both high temperature and a flow regime with increased residence time of the desorbed plume in the desolvation region promote the generation of multiply charged ions. Without such measures the application of an electric ion extraction field significantly increases the ion signal intensity of singly charged species while the detection of multiply charged species is less dependent on the extraction field. In general, optimization of high temperature application facilitates the predominant formation and detection of multiply charged compared to singly charged ion species. In this study an experimental set-up and optimization strategy is described for liquid AP-MALDI MS which improves the ionization efficiency of selected ion species up to 14 times. In combination with ion mobility separation, the method allows the detection of multiply charged peptide and protein ions for analyte solution concentrations as low as 2fmol/μL (0.5μL, i.e. 1fmol, deposited on the target) with very low sample consumption in the low nL-range.
Different phosphoinositides are synthesized in cell membranes in order to perform a variety of functions. One of the most abundant of these lipids is phosphatidylinositol (PI) 4-phosphate (PI4P), ...which is formed in human eukaryotes by type II and type III phosphatidylinositol 4-kinase (PI4K II and III) activities. PI4K II activity occurs in many different subcellular membranes, although no detailed analysis of the distribution of this activity has been reported. Using density gradient ultracentrifugation, we have previously found that in A431 cells the predominant PI4K activity arises from a type II alpha enzyme that is localized to a buoyant membrane fraction of unknown origin Waugh, Lawson, Tan and Hsuan (1998) J. Biol. Chem. 273, 17115-17121. We show here that these buoyant membranes contain an activated form of PI4K II alpha that can be separated from the bulk of the PI4K II alpha protein in A431 and COS-7 cells. Proteomic analysis revealed that the buoyant membrane fraction contains numerous endoplasmic reticulum (ER)-marker proteins, although it was separated from the bulk of the ER, ER-Golgi intermediate compartment, transitional ER, Golgi and other major subcellular membranes. Furthermore, the majority of the cytoplasmic valosin-containing protein (VCP), an AAA+ATPase implicated in various ER, transitional ER, Golgi and nuclear functions, was almost completely localized to the same buoyant membrane fraction. Co-localization of VCP and PI4K activity was confirmed by co-immunoprecipitation. These results suggest the previously unsuspected existence of an ER-related domain in which the bulk of the cellular PI4P synthesis and VCP are localized.
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•First-time demonstration of an LC-MALDI MS/MS bottom-up proteomics workflow utilizing multiply charged precursor ions.•The developed (nanoUHP)LC-MALDI method is compatible with the ...use of TFA in the LC mobile phase.•Off-line analysis and extremely low sample consumption allow for multiple re-analyses of the same sample.•Storage of MALDI samples on fully spotted target plates is possible for months without significant sample degradation.
Liquid AP-MALDI can produce predominantly multiply charged ESI-like ions and stable durable analyte ion yields with samples allowing good shot-to-shot reproducibility and exhibiting self-healing properties during laser irradiation. In this study, LC-MALDI MS/MS workflows that utilize multiply charged ions are reported for the first time and compared with standard LC-ESI MS/MS for bottom-up proteomic analysis. The proposed method is compatible with trifluoroacetic acid as an LC ion pairing reagent and allows multiple MS/MS acquisitions of the LC-separated samples without substantial sample consumption. In addition, the method facilitates the storage of fully spotted MALDI target plates for months without significant sample degradation.
•Used MALDI imaging to view the distribution of proteins in malignant penile tissue.•Found heterogeneous and tissue-specific distribution of a number of molecular species.•Verified that S100A4 is ...differentially expressed in malignant epithelial cells.•Obtained significant correlation between S100A4 positive cells and grade of tumour.
MS-based proteomic methods were utilised for the first time in the discovery of novel penile cancer biomarkers. MALDI MS imaging was used to obtain the in situ biomolecular MS profile of squamous cell carcinoma of the penis which was then compared to benign epithelial MS profiles. Spectra from cancerous and benign tissue areas were examined to identify MS peaks that best distinguished normal epithelial cells from invasive squamous epithelial cells, providing crucial evidence to suggest S100A4 to be differentially expressed. Verification by immunohistochemistry resulted in positive staining for S100A4 in a sub-population of invasive but not benign epithelial cells.
Rationale
Liquid atmospheric pressure matrix‐assisted laser desorption/ionisation (AP‐MALDI) has been shown to enable the production of electrospray ionisation (ESI)‐like multiply charged analyte ...ions with little sample consumption and long‐lasting, robust ion yield for sensitive analysis by mass spectrometry (MS). Previous reports have focused on positive ion production. Here, we report an initial optimisation of liquid AP‐MALDI for ESI‐like negative ion production and its application to the analysis of peptides/proteins, DNA and lipids.
Methods
The instrumentation employed for this study is identical to that of earlier liquid AP‐MALDI MS studies for positive analyte ion production with a simple non‐commercial AP ion source that is attached to a Waters Synapt G2‐Si mass spectrometer and incorporates a heated ion transfer tube. The preparation of liquid MALDI matrices is similar to positive ion mode analysis but has been adjusted for negative ion mode by changing the chromophore to 3‐aminoquinoline and 9‐aminoacridine for further improvements.
Results
For DNA, liquid AP‐MALDI MS analysis benefited from switching to 9‐aminoacridine‐based MALDI samples and the negative ion mode, increasing the number of charges by up to a factor of 2 and the analyte ion signal intensities by more than 10‐fold compared with the positive ion mode. The limit of detection was recorded at around 10 fmol for ATGCAT. For lipids, negative ion mode analysis provided a fully orthogonal set of detected lipids.
Conclusions
Negative ion mode is a sensitive alternative to positive ion mode in liquid AP‐MALDI MS analysis. In particular, the analysis of lipids and DNA benefited from the complementarity of the detected lipid species and the vastly greater DNA ion signal intensities in negative ion mode.
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