An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture

• DOI: 10.1074/mcp.m800190-mcp200
• Publication Date: 2008-10-21T00:14:30Z
• Authors: Sean C. Bendall; Chris Hughes; J. Larry Campbell; Morag H. Stewart; Paula Pittock; Suya Liu; Eric Bonneil; Pierre Thibault; Mickie Bhatia; Gilles A. Lajoie
• Keywords: Orbitrap; Proteome

The derivation and long-term maintenance of human embryonic stem cells (hESCs) has been established in culture formats that are both dependent independent support (feeder) cells. However, the factors responsible for preserving viability hESCs a nascent state remain unknown. We describe mass spectrometry-based method probing secretome hESC microenvironment to identify potential regulating protein low abundance. Individual samples were analyzed several times, using successive (m/z) retention time-directed exclusion, without sampling same peptide ion twice. This iterative exclusion -mass spectrometry (IE-MS) approach more than doubled metrics comparison simple repeat analysis on instrument, even after extensive sample pre-fractionation. Furthermore, implementation IE-MS was shown enhance performance an older quadrupole time flight (Q-ToF) MS. resulting number identified peptides approached parallel newer LTQ-Orbitrap combination results instruments proved be superior achieved by single instrument identification additional proteins. Using strategy, combined with complementary gel- solution-based fractionation methods, extensively probed. Over 10 12 times extracellular proteins observed compared previously published surveys. detection undetectable growth factors, present at concentrations ranging from 10(-9) 10(-11) g/ml, highlights depth our profiling. provides reliable technique greatly enhances increasing effective MS-based proteomic should widely applicable any LC-MS/MS platform or biological system.