Coupled Global and Targeted Proteomics of Human Embryonic Stem Cells during Induced Differentiation

Abstract

Elucidating the complex combinations of growth factors and signaling molecules that maintain pluripotency or, alternatively, promote the controlled differentiation of human embryonic stem cells (hESCs) has important implications for the fundamental understanding of human development, devising cell replacement therapies, and cancer cell biology. hESCs are commonly grown on irradiated mouse embryonic fibroblasts (MEFs) or in conditioned medium from MEFs. These culture conditions interfere with many experimental conclusions and limit the ability to perform conclusive proteomics studies. The current investigation avoided the use of MEFs or MEF-conditioned medium for hESC culture, allowing global proteomics analysis without these confounding conditions, and elucidated neural cell-specific signaling pathways involved in noggin-induced hESC differentiation. Based on these analyses, we propose the following early markers of hESC neural differentiation: collapsin response mediator proteins 2 and 4 and the nuclear autoantigenic sperm protein as a marker of pluripotent hESCs. We then developed a directed mass spectrometry assay using multiple reaction monitoring (MRM) to identify and quantify these markers and in addition the epidermal ectoderm marker cytokeratin-8. Analysis of global proteomics, quantitative RT-PCR, and MRM data led to testing the isoform interference hypothesis where redundant peptides dilute quantification measurements of homologous proteins. These results show that targeted MRM analysis on nonredundant peptides provides more exact quantification of homologous proteins. This study describes the facile transition from discovery proteomics to targeted MRM analysis and allowed us to identify and verify several potential biomarkers for hESCs during noggin-induced neural and BMP4-induced epidermal ectoderm differentiation. Molecular & Cellular Proteomics 7:750 -767, 2008.Human embryonic stem cells (hESCs) 1 are perhaps the most promising source of cells for regenerative medicine and treatment of disease. Despite extensive research aimed at elucidating and controlling the processes of self-renewal and lineage-specific differentiation, much remains to be learned regarding the basic cell biology of hESCs before their clinical potential can be realized. Our current knowledge regarding the complex regulation of lineage segregation during development arises primarily from in vivo investigations in invertebrate and mouse. Furthermore manipulation of the signaling pathways initiating and controlling lineage differentiation during development, including the role of bone morphogenic proteins (BMPs), has been expedited by in vitro studies of mouse embryonic stem cells (mESCs). Only recently has considerable research commenced on hESCs.BMPs were originally characterized based on their ability to induce cartilage and bone formation. They are now known to be multifunctional regulators of development, including many non-osteogenic processes (1). There are as many as 30 different BMP family members classified according to...

Current affairs in quantitative targeted proteomics: multiple reaction monitoring-mass spectrometry

“…Whether or not empirical data has been collected, it is recommended that MRM transitions are designed for chosen peptides approximately conforming to the following rules [1,12]: (i) With electrospray ionization, doubly charged parent ions are preferred over singly charged for Q1, however a triply charged parent ion mass is assumed if the peptide sequence contains a histidine residue and can be used if the peptide has a mass greater than 1500 Da. (ii) y-ions are most often preferred over b-ions to ensure the transition contains a labeling site.…”

“…This is an area where MRM-MS is really gaining interest and momentum. Not only can a targeted approach such as MRM-MS lessen the ever challenging dynamic range issues most commonly encountered during global proteomics experiments thereby digging deeper into the low abundance proteome and low stochiometric PTM, this methodology is showing tremendous utility in the verification of both global proteomics identification and quantification data [12,23,[35][36][37][38][39]. Unfortunately, global proteomics data can be inconclusive.…”

“…Specificity can be achieved by the selection of sequences with no homology in BLAST, as well chromatographic retention standardization. MRM methodologies are particularly useful for measurement of highly homologous protein isoforms because of the high level of specificity achieved [11,12]. Single amino-acid changes in a peptide sequence can often be distinguished by MRM enabling resolution and quantification of closely related isoforms.…”

“…Additionally, chemical labeling of synthetic peptides with the ICAT reagents has been used to create internal standards for accurate quantitation of P450 proteins [11]. Labeling cell lysates with iTRAQ reagents to determine relative differential regulation of proteins has been completed as well [12]. Using the isobaric and/or non-isobaric chemical labeling reagents, one can create unique and individualized internal standards by labeling one cell state with a light reagent which then acts as a control.The other cell state to be quantified relative to the control is labeled with the heavy reagent.…”

Embryonic stem cell application in drug discovery

“…Similarly, protein interactions can be mapped in ES cells to enable systematic discovery of regulatory pathways [47,48] . Indeed, a method for defining the proteome of stem cell populations using isobaric tags for relative and absolute quantitation has been developed.…”