TY - JOUR
T1 - Toward Analysis of Proteins in Single Cells
T2 - A Quantitative Approach Employing Isobaric Tags with MALDI Mass Spectrometry Realized with a Microfluidic Platform
AU - Yang, Mian
AU - Nelson, Randall
AU - Ros, Alexandra
N1 - Publisher Copyright:
© 2016 American Chemical Society.
PY - 2016/7/5
Y1 - 2016/7/5
N2 - Protein identification and quantification in individual cells is essential to understand biological processes such as those involved in cell apoptosis, cancer, biomarker discovery, disease diagnostics, pathology, or therapy. Compared with present single cell genome analysis, probing the protein content of single cells has been hampered by the lack of a protein amplification technique. Here, we report the development of a quantitative mass spectrometric approach combined with microfluidic technology reaching the detection sensitivity of high abundant proteins in single cells. A microfluidic platform with a series of chambers and valves, ensuring a set of defined wells for absolute quantification of targeted proteins, was developed and combined with isotopic labeling strategies employing isobaric tags for relative and absolute quantitation (iTRAQ)-labels. To this aim, we adapted iTRAQ labeling to an on-chip protocol. Simultaneous protein digestion and labeling performed on the microfluidic platform rendered the labeling strategy compatible with all necessary manipulation steps on-chip, including the matrix delivery for MALDI-TOF analysis. We demonstrate this approach with the apoptosis related protein Bcl-2 and quantitatively assess the number of Bcl-2 molecules detected. We anticipate that this approach will eventually allow quantification of protein expression on the single cell level.
AB - Protein identification and quantification in individual cells is essential to understand biological processes such as those involved in cell apoptosis, cancer, biomarker discovery, disease diagnostics, pathology, or therapy. Compared with present single cell genome analysis, probing the protein content of single cells has been hampered by the lack of a protein amplification technique. Here, we report the development of a quantitative mass spectrometric approach combined with microfluidic technology reaching the detection sensitivity of high abundant proteins in single cells. A microfluidic platform with a series of chambers and valves, ensuring a set of defined wells for absolute quantification of targeted proteins, was developed and combined with isotopic labeling strategies employing isobaric tags for relative and absolute quantitation (iTRAQ)-labels. To this aim, we adapted iTRAQ labeling to an on-chip protocol. Simultaneous protein digestion and labeling performed on the microfluidic platform rendered the labeling strategy compatible with all necessary manipulation steps on-chip, including the matrix delivery for MALDI-TOF analysis. We demonstrate this approach with the apoptosis related protein Bcl-2 and quantitatively assess the number of Bcl-2 molecules detected. We anticipate that this approach will eventually allow quantification of protein expression on the single cell level.
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U2 - 10.1021/acs.analchem.5b03419
DO - 10.1021/acs.analchem.5b03419
M3 - Article
AN - SCOPUS:84977628124
SN - 0003-2700
VL - 88
SP - 6672
EP - 6679
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 13
ER -