Proteomic Analysis of Lipid Microdomains from Lipopolysaccharide-Activated Human Endothelial Cells

The endothelium plays a critical role in orchestrating the inflammatory response seen during sepsis. Many of the inflammatory effects of Gram-negative sepsis are elicited by lipopolysaccharide (LPS), a glycolipid component of bacterial cell walls. Lipid-rich microdomains have been shown to concentrate components of the LPS signaling system. However, much remains to be learned about which proteins are constituents of lipid microdomains, and how these are regulated following cell activation. Progress in this area would be accelerated by employing global proteomic analyses, but the hydrophobicity of membrane proteins presents an analytical barrier to the effective application of such approaches. Herein, we describe a method to isolate detergent-resistant membranes from endothelial cells, and prepare these samples for proteomic analysis in a way that is compatible with subsequent separations and mass spectrometric (MS) analysis. In the application of these sample preparation and MS analyses, 358 proteins from the lipid-rich microdomains of LPS-activated endothelial cell membranes have been identified of which half are classified as membrane proteins by Gene Ontology. We also demonstrate that the sample preparation method used for solubilization and trypsin digestion of lipid-rich microdomains renders the membrane spanning sequences of transmembrane proteins accessible for endoproteolytic hydrolysis. This analysis sets the analytical foundation for an in-depth probing of LPS signaling in endothelial cells. Keywords: lipopolysaccharide • endothelium • caveolae • lipid rafts • lipid-rich microdomains • inflammatio

Probing Early Growth Response 1 Interacting Proteins at the Active Promoter in Osteoblast Cells Using Oligoprecipitation and Mass Spectrometry

Current advances in proteomics have allowed for a rapidly expanding integration of associated methodologies with more traditional molecular and biochemical approaches to the study of cell function. Recent studies on the role of inorganic phosphate have suggested this ion is a novel signaling molecule capable of altering the function of numerous cell types. Elevated inorganic phosphate generated in the extracellular microenvironment by differentiating osteoblasts has recently been determined to act through a largely uncharacterized mechanism as an important signaling molecule responsible for altering the transcription of various genes during osteoblast differentiation. The transcription factor, early growth response protein 1 (EGR1), has previously been shown to be involved in the early response of osteoblasts to inorganic phosphate. To elucidate the role of EGR1 as a potential early regulator of transcription in the inorganic phosphate response, an oligoprecipitation procedure was optimized to capture the DNA bound, transcriptionally active form of EGR1. The interacting proteins thusly captured were identified using mass spectrometry (MS). Proteins involved in transcription, RNA processing, and chromatin modification were identified by this approach. The combined oligoprecipitation-MS approach presented here is highly effective for isolating and characterizing entire transcriptional complexes in the DNA bound state and is broadly extendable to the identification of both known and unknown transcription factor protein complexes. Keywords: oligoprecipitation • EGR1 • DNA-protein interaction • mass spectrometry • inorganic phosphat

Identification of the SELDI ProteinChip Human Serum Retentate by Microcapillary Liquid Chromatography-Tandem Mass Spectrometry

Surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry (MS) has been widely applied for conducting biomarker research with the goal of discovering patterns of proteins and/or peptides from biological samples that reflect disease status. Many diseases, ranging from cancers of the colon, breast, and prostate to Alzheimer's disease, have been studied through serum protein profiling using SELDI-based methods. Although the results from SELDI-based diagnostic studies have generated a great deal of excitement and skepticism alike, the basis of the molecular identities of the features that underpin the diagnostic potential of the mass spectra is still largely unexplored. A detailed investigation has been undertaken to identify the compliment of serum proteins that bind to the commonly used weak cation exchange (WCX-2) SELDI protein chip. Following incubation and washing of a standard serum sample on the WCX-2 sorbent, proteins were harvested, digested with trypsin, fractionated by strong cation exchange liquid chromatography (LC), and subsequently analyzed by microcapillary reversed-phase LC coupled online with an ion-trap mass spectrometer. This analysis resulted in the identification of 383 unique proteins in the WCX-2 serum retentate. Among the proteins identified, 50 (13%) are documented clinical biomarkers with 36 of these (72%) identified from multiple peptides. Keywords: SELDI • biomarker • serum proteomics • multidimensional fractionation • mass spectrometr

Identification of the SELDI ProteinChip Human Serum Retentate by Microcapillary Liquid Chromatography-Tandem Mass Spectrometry

Surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry (MS) has been widely applied for conducting biomarker research with the goal of discovering patterns of proteins and/or peptides from biological samples that reflect disease status. Many diseases, ranging from cancers of the colon, breast, and prostate to Alzheimer's disease, have been studied through serum protein profiling using SELDI-based methods. Although the results from SELDI-based diagnostic studies have generated a great deal of excitement and skepticism alike, the basis of the molecular identities of the features that underpin the diagnostic potential of the mass spectra is still largely unexplored. A detailed investigation has been undertaken to identify the compliment of serum proteins that bind to the commonly used weak cation exchange (WCX-2) SELDI protein chip. Following incubation and washing of a standard serum sample on the WCX-2 sorbent, proteins were harvested, digested with trypsin, fractionated by strong cation exchange liquid chromatography (LC), and subsequently analyzed by microcapillary reversed-phase LC coupled online with an ion-trap mass spectrometer. This analysis resulted in the identification of 383 unique proteins in the WCX-2 serum retentate. Among the proteins identified, 50 (13%) are documented clinical biomarkers with 36 of these (72%) identified from multiple peptides. Keywords: SELDI • biomarker • serum proteomics • multidimensional fractionation • mass spectrometr

Investigation of the Mouse Serum Proteome

With the rapid assimilation of genomic information and the equally impressive developments in the field of proteomics, there is an unprecedented interest in biomarker discovery. Although human biofluids represent increasingly attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. One of the most extensively used animal models for studying human disease is mouse because, unlike humans, they represent a highly controllable experimental model system. Unfortunately, very little is known about the proteomic composition of mouse serum. In this study, a multidimensional fractionation approach on both the protein and the peptide level that does not require depletion of highly abundant serum proteins was combined with tandem mass spectrometry to characterize proteins within mouse serum. Over 12 300 unique peptides that originate from 4567 unique proteinsapproximately 16% of all known mouse proteinswere identified. The results presented here represent the broadest proteome coverage in mouse serum and provide a foundation from which quantitative comparisons can be made in this important animal model. Keywords: mouse • serum proteomics • multidimensional fractionation • tandem mass spectrometr

Investigation of the Mouse Serum Proteome

With the rapid assimilation of genomic information and the equally impressive developments in the field of proteomics, there is an unprecedented interest in biomarker discovery. Although human biofluids represent increasingly attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. One of the most extensively used animal models for studying human disease is mouse because, unlike humans, they represent a highly controllable experimental model system. Unfortunately, very little is known about the proteomic composition of mouse serum. In this study, a multidimensional fractionation approach on both the protein and the peptide level that does not require depletion of highly abundant serum proteins was combined with tandem mass spectrometry to characterize proteins within mouse serum. Over 12 300 unique peptides that originate from 4567 unique proteinsapproximately 16% of all known mouse proteinswere identified. The results presented here represent the broadest proteome coverage in mouse serum and provide a foundation from which quantitative comparisons can be made in this important animal model. Keywords: mouse • serum proteomics • multidimensional fractionation • tandem mass spectrometr

Combined Chemical and Enzymatic Stable Isotope Labeling for Quantitative Profiling of Detergent-Insoluble Membrane Proteins Isolated Using Triton X-100 and Brij-96

Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five noncysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content. Keywords: quantitative proteomics • combined 16O/18O and ICAT stable isotopic labeling • Triton X-100 and Brij-96 detergent-insoluble membrane protein

Combined Chemical and Enzymatic Stable Isotope Labeling for Quantitative Profiling of Detergent-Insoluble Membrane Proteins Isolated Using Triton X-100 and Brij-96

Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five noncysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content. Keywords: quantitative proteomics • combined 16O/18O and ICAT stable isotopic labeling • Triton X-100 and Brij-96 detergent-insoluble membrane protein

Investigation of the Mouse Serum Proteome

With the rapid assimilation of genomic information and the equally impressive developments in the field of proteomics, there is an unprecedented interest in biomarker discovery. Although human biofluids represent increasingly attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. One of the most extensively used animal models for studying human disease is mouse because, unlike humans, they represent a highly controllable experimental model system. Unfortunately, very little is known about the proteomic composition of mouse serum. In this study, a multidimensional fractionation approach on both the protein and the peptide level that does not require depletion of highly abundant serum proteins was combined with tandem mass spectrometry to characterize proteins within mouse serum. Over 12 300 unique peptides that originate from 4567 unique proteinsapproximately 16% of all known mouse proteinswere identified. The results presented here represent the broadest proteome coverage in mouse serum and provide a foundation from which quantitative comparisons can be made in this important animal model. Keywords: mouse • serum proteomics • multidimensional fractionation • tandem mass spectrometr

Investigation of the Mouse Serum Proteome

With the rapid assimilation of genomic information and the equally impressive developments in the field of proteomics, there is an unprecedented interest in biomarker discovery. Although human biofluids represent increasingly attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. One of the most extensively used animal models for studying human disease is mouse because, unlike humans, they represent a highly controllable experimental model system. Unfortunately, very little is known about the proteomic composition of mouse serum. In this study, a multidimensional fractionation approach on both the protein and the peptide level that does not require depletion of highly abundant serum proteins was combined with tandem mass spectrometry to characterize proteins within mouse serum. Over 12 300 unique peptides that originate from 4567 unique proteinsapproximately 16% of all known mouse proteinswere identified. The results presented here represent the broadest proteome coverage in mouse serum and provide a foundation from which quantitative comparisons can be made in this important animal model. Keywords: mouse • serum proteomics • multidimensional fractionation • tandem mass spectrometr