Rare Cell Proteomic Reactor Applied to Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics Study of Human Embryonic Stem Cell Differentiation

Abstract

The molecular basis governing the differentiation of human embryonic stem cells (hESCs) remains largely unknown. Systems-level analysis by proteomics provides a unique approach to tackle this question. However, the requirement of a large number of cells for proteomics analysis (i.e. 10 6 -10 7 cells) makes this assay challenging, especially for the study of rare events during hESCs lineage specification. Here, a fully integrated proteomics sample processing and analysis platform, termed rare cell proteomic reactor (RCPR), was developed for large scale quantitative proteomics analysis of hESCs with ϳ50,000 cells. hESCs were completely extracted by a defined lysis buffer, and all of the proteomics sample processing procedures, including protein preconcentration, reduction, alkylation, and digestion, were integrated into one single capillary column with a strong cation exchange monolith matrix. Furthermore, on-line two-dimensional LC-MS/MS analysis was performed directly using RCPR as the first dimension strong cation exchange column. 2,281 unique proteins were identified on this system using only 50,000 hESCs. For stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative study, a ready-to-use and chemically defined medium and an in situ differentiation procedure were developed for complete SILAC labeling of hESCs with well characterized self-renewal and differentiation properties. Mesoderm-enriched differentiation was studied by RCPR using 50,000 hESCs, and 1,086 proteins were quantified with a minimum of two peptides per protein. Of these, 56 proteins exhibited significant changes during mesoderm-enriched differentiation, and eight proteins were demonstrated for the first time to be overexpressed during early mesoderm development. This work provides a new platform for the study of rare cells and in particular for further elucidating proteins that govern the mesoderm lineage specification of human pluripotent stem cells.

Critical Role of Endoplasmic Reticulum Aminopeptidase 1 in Determining the Length and Sequence of Peptides Bound and Presented by HLA–B27

“…cells with different arginine isotopes (using a SILACbased proteomics approach), and then pooled the samples for HLA-B27 peptide isolation and MS/MS analysis (24,25). Arginine was selected for SILAC isotope labeling because it is a dominant "anchor" amino acid at position 2 (P2) of HLA-B27 peptide ligands (26-28), whereas it is not an anchor residue for the other alleles expressed (HLA-Cw*04 and HLA-B*35), albeit at low levels, in C1R cells (29)(30)(31).…”

An Integrated Platform for Isolation, Processing, and Mass Spectrometry-based Proteomic Profiling of Rare Cells in Whole Blood*

“…Several approaches such as denaturant-assisted lysis, acetone precipitation, filter-aided sample preparation, and monolithic microreactor-based techniques have been developed for processing small amounts of sample, for example 500 -1000 cultured cells (15)(16)(17). However, these methodologies only allow identification of a few hundred proteins at these levels.…”

Online Peptide Fractionation Using a Multiphasic Microfluidic Liquid Chromatography Chip Improves Reproducibility and Detection Limits for Quantitation in Discovery and Targeted Proteomics*

“…Because of the small dimension of these devices, they are readily able to integrate into nanoLC workflows. Various applications have been described including increasing proteome coverage (22,27,28) and targeting of phosphopeptides (24,31,32), glycopeptides and released glycans (29,33,34).…”