Abstract
The derivation and long-term maintenance of human embryonic stem cells (hESCs) has been established in culture formats that are both dependent and independent of support (feeder) cells. However, the factors responsible for preserving the viability of hESCs in a nascent state remain unknown. We describe a mass spectrometry-based method for probing the secretome of the hESC culture microenvironment to identify potential regulating protein factors that are in low abundance. Individual samples were analyzed several times, using successive mass (m/z) and retention time-directed exclusion, without sampling the same peptide ion twice. This iterative exclusion -mass spectrometry (IE-MS) approach more than doubled protein and peptide metrics in comparison to a simple repeat analysis method on the same instrument, even after extensive sample pre-fractionation. Furthermore, implementation of the IE-MS approach was shown to enhance the performance of an older quadrupole time of flight (Q-ToF) MS. The resulting number of identified peptides approached that of a parallel repeat analysis on a newer LTQ-Orbitrap MS. The combination of the results of both instruments proved to be superior to that achieved by a single instrument in the identification of additional proteins. Using the IE-MS strategy, combined with complementary gel- and solution-based fractionation methods, the hESC culture microenvironment was extensively probed. Over 10 to 12 times more extracellular proteins were observed compared with previously published surveys. The detection of previously undetectable growth factors, present at concentrations ranging from 10(-9) to 10(-11) g/ml, highlights the depth of our profiling. The IE-MS approach provides a simple and reliable technique that greatly enhances instrument performance by increasing the effective depth of MS-based proteomic profiling. This approach should be widely applicable to any LC-MS/MS instrument platform or biological system.
Highlights
The derivation and long-term maintenance of human embryonic stem cells has been established in culture formats that are both dependent and independent of support cells
Previous genomic-based studies suggested the presence of such networks of human embryonic stem cells (hESCs) transcriptional regulation [7]; these networks were not correlated to the extracellular microenvironment that controls hESC fate
In the MS analysis of a complex biological sample, even if only a small portion (ϳ10%) of these peptides were observable/present and they were further reduced in complexity 10-fold through fractionation, ϳ6 ϫ 104 uniquely observable m/z species would still be in each MS analysis
Summary
The derivation and long-term maintenance of human embryonic stem cells (hESCs) has been established in culture formats that are both dependent and independent of support (feeder) cells. We describe a mass spectrometrybased method for probing the secretome of the hESC culture microenvironment to identify potential regulating protein factors that are in low abundance. In a typical LC-MS/MS experiment in data-dependent acquisition (DDA) mode, the most abundant peptides ions are selected preferentially for MS/MS fragmentation, resulting in the identification of the most abundant proteins in a given mixture To overcome this limitation, a number of strategies have been developed. These include organellar separation [18], as well as several pre-fractionation and enrichment strategies [19] Despite these enhancements, many proteins remain unidentified, because the dynamic range of the experiment is reduced by the vast excess of peptides from high-abundance proteins present. This is further compounded by the intrinsic limitation of duty cycle and dynamic range of mass spectrometry instrumentation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
