Immobilized pH gradient-driven paper-based IEF: a new method for fractionating complex peptide mixtures before MS analysis.

INTRODUCTION: The vast difference in the abundance of different proteins in biological samples limits the determination of the complete proteome of a cell type, requiring fractionation of proteins and peptides before MS analysis. METHODS: We present a method consisting of electrophoresis of complex mixtures of peptides using a strip of filter paper cut into 20 sections laid end to end over a 24-cm-long IPG strip, the pH gradient of which would drive the electrophoresis. Peptides absorbed onto individual paper pads after electrophoresis are subsequently recovered into a buffer solution, thus dividing a complex peptide mixture according to pI into 20 liquid fractions. This paper-based IEF method (PIEF) was compared side-by-side with a similar but liquid-based Offgel electrophoresis (OGE) by analyzing iTRAQ-labeled peptide mixtures of membrane proteins from four different cell types. RESULTS: PIEF outperformed OGE in resolving acidic peptides, whereas OGE did a better job in recovering relatively basic peptides. OGE and PIEF were quite comparable in their coverage, identifying almost equal number of distinct proteins (PIEF =1174; OGE = 1080). Interestingly, however, only 675 were identified by both of them, each method identifying many unique proteins (PIEF = 499; OGE = 415). Thus, the two methods uncovered almost 40% more proteins compared to what is obtained by only one method. CONCLUSION: This initial investigation demonstrates the technical feasibility of PIEF for complementing OGE. PIEF uses standard IPG IEF equipment, requires no specialized apparatus (e.g., OGE fractionator) and may be integrated into peptide mapping strategies for clinical samples.

Proteome profiling of aging in mouse models: differential expression of proteins involved in metabolism, transport, and stress response in kidney.

Aging is a time-dependent complex biological phenomenon observed in various organs and organelles of all living organisms. To understand the molecular mechanism of age-associated functional loss in aging kidneys, we have analyzed the expression of proteins in the kidneys of young (19-22 wk) and old (24 months) C57/BL6 male mice using 2-DE followed by LC-MS/MS. We found that expression levels of 49 proteins were upregulated (p < or = 0.05), while that of only ten proteins were downregulated (p < or = 0.05) due to aging. The proteins identified belong to three broad functional categories: (i) metabolism (e.g., aldehyde dehydrogenase family, ATP synthase beta-subunit, malate dehydrogenase, NADH dehydrogenase (ubiquinone), hydroxy acid oxidase 2), (ii) transport (e.g., transferrin), and (iii) chaperone/stress response (e.g., Ig-binding protein, low density lipoprotein receptor-related protein associated protein 1, selenium-binding proteins (SBPs)). Some proteins with unknown functions were also identified as being differentially expressed. ATP synthase beta subunit, transferrin, fumarate hydratase, SBPs, and albumin are present in multiple forms, possibly arising due to proteolysis or PTMs. The above functional categories suggest specific mechanisms and pathways for age-related kidney degeneration.

Gene expression analysis at the single cell level using the human bone marrow stromal cell as a model: sample preparation methods.

Recent advances in molecular technology, including gene expression microarray analysis, have allowed researchers to examine global patterns of gene expression at high resolution in populations of cultured cells or tissues. Although these techniques can be applied with great sophistication and are useful for address ing many biological questions in cell populations, it is also of great value to assess gene expression at the level of the single cell. This can be achieved by one of two different approaches: (1) specific cell types can be purified from heterogeneous tissues or cultures using immunological methods such as fluorescence-based or magnetic cell sorting or laser capture microdissection, followed by amplification of target cell nucleic acids, and analysis of expressed genes; or (2) immunohisto-chemical studies and in situ expression studies on identical tissue sections can be used to identify genes or sets of genes whose expression correlates with a morpho logically or immunochemically distinct cell-type. Using either approach, the target cell types are identified by their morphological or immunohistochemical properties. This chapter is a primer on using single cell gene expression technology to study human bone marrow stromal cells that express mixed lineage markers. Cytomorphological, cytochemical, and immunocytochemical methods as well as gene expression microarray studies demonstrated that single stromal cells simulta neously express markers associated with osteoblast, fibroblast, muscle, and adi-pocyte differentiation, suggesting that these stromal cells are mesenchymal progenitor cells that have multilineage differentiation capacity. These data charac terize human bone marrow stromal cells as adult stem cells. Because of their pluripotent nature, single cell gene expression technology is particularly critical for characterizing and developing the therapeutic potential of these cells.

Proteomics strategy based on liquid-phase IEF and 2-D DIGE: application to bone marrow mesenchymal progenitor cells.

Global comparative proteomics is a promising new approach with broad application in basic and clinical biological science. Recent advances include the development of 2-D DIGE, a proteomic equivalent to mRNA differential display, in which differentially labeled samples are multiplexed and analyzed by high-resolution 2-DE. This study presents a new 2-D DIGE protocol, in which complex protein samples from normal and leukemic human bone marrow mesenchymal progenitor cells were used as model samples for a novel combination of liquid-phase IEF with 2-D DIGE. Using liquid-phase IEF, the normal and leukemic cells were pre-fractionated into five subproteomes after multiplexing but prior to DIGE. Under these conditions, 2-D DIGE resolved >5000 protein-containing spots within the pH range 4.6-7.0. Differential labeling combined with subsequent MALDI-MS/MS identified proteins that were differentially expressed in leukemic cells. This analysis mapped protein identities to 128 mesenchymal progenitor cell proteins with at least one unique peptide match at >95% confidence. Of these proteins, 72 (56%) were expressed more than 1.25-fold higher or lower in leukemic cells compared with normal cells (p<0.05). These data were used to infer gene ontology biological processes that may be altered in leukemic bone marrow mesenchymal progenitor cells.

An integrated approach to mapping the proteome of the human bone marrow stromal cell.

Bone marrow stromal cells, also known as mesenchymal stem or progenitor cells, are thought to play a critical role in hematopoiesis. Because hematopoiesis is dysregulated in leukemic individuals, expression profiling in leukemic stromal cells might yield valuable insights into mechanisms of normal and dysregulated hematopoiesis. Here, proteomics was used to identify expressed proteins in normal and leukemic bone marrow stromal cells. Isobaric tags for relative and absolute quantification (iTRAQ) technology and MS/MS were used to identify and quantify proteins in bone marrow stromal cells from two normal and two leukemic individuals. Approximately 900 distinct proteins were identified with >95% confidence, 73 of which were differentially expressed in leukemic stromal cells. cDNA microarray analysis on Affymetrix oligonucleotide gene chips showed that transcripts correlated with approximately 90% of the expressed proteins. A novel integrated approach for mining and visualization of iTRAQ data is presented. The results provide an initial assessment of the proteome in human bone marrow stromal cells, laying the groundwork for comprehensive analysis of the proteome in these cells, and ultimately, for improved understanding of normal and dysregulated hematopoiesis.

Multilineage gene expression in human bone marrow stromal cells as evidenced by single-cell microarray analysis.

The nonhematopoietic stromal cells of the bone marrow are critical for the development of hematopoietic stem cells into functionally competent blood cells. This study addresses the question of whether bone marrow stromal cell cultures in the Dexter system propagate multiple different mesenchymal stromal cell types or one stromal cell type that expresses multiple phenotypes. Results show that isolated single stromal cells simultaneously express transcripts associated with osteoblast, fibroblast, muscle, and adipocyte differentiation. Furthermore, isolated single stromal cells simultaneously express transcripts characteristic of epithelial cells, endothelial cells, and neural/glial cells. Isolated single stromal cells also express transcripts for CD45, CD19, CD10, CD79a, and representative proto-oncogenes and transcription factors, which are typically associated with normal and neoplastic hematopoietic cells. These findings suggest that the nonhematopoietic mesenchymal cells and the hematopoietic B-lymphocytes have a common progenitor. This is consistent with the idea that progenitor cells express genes that are characteristic of the multiple lineage paths that such cells may be capable of adopting. This study demonstrates the technical feasibility of transcriptome analysis of individual primary cell-culture grown stromal cells and supports the concept that bone marrow stromal cells are relatively homogeneous and show a phenotypic signature of potential multilineage differentiation capacity.