Institute of Biomedical Sciences
High pH Fractionation for Enhancing the Proteome Coverage in Tissues Dissected from the Early Frog (Xenopus laevis) Embryo
Poster Number
7
Document Type
Poster
Keywords
Proteomics, Developmental Biology, Xenopus laevis, LC-MS,
Publication Date
4-2017
Abstract
Discovery measurement of the proteome raises an opportunity to better understand how differential expression of the genome underlies the formation of various tissue types during early embryonic development. To enhance the detectable proteome, we and others have developed high-sensitivity mass spectrometry instruments and protocols for the removal of abundant proteins, such as pelleting of yolk proteins using density gradients in the frog (Xenopus laevis) embryo. Here, we test high pH fractionation to improve the identification and quantification of proteins in the early developing X. laevis embryo.
The main goal of this work was to enhance the identification and quantification of proteins in the early developing frog (Xenopus laevis) embryo. While X. laevis is a powerful model of developmental biology and health studies, the proteome of the early embryo is dominated by abundant yolk proteins (>90% of the proteome), which hinders the characterization of low-abundance proteins. Building on the success of high pH fractionation, we proposed that this approach would help improve protein identifications in Xenopus tissues by minimizing the complexity of the samples prior to liquid chromatography-Mass spectrometry analysis. To test this, we pooled tissues from n = 10 embryos and extracted proteins using a SDS based lysis buffer and precipitated the proteins overnight in cold acetone. The extracted proteins were then digested with trypsin for 5 h at 37 ºC. The resulting peptides were split into two sections: one was processed further by high-pH fractionation and the other was measured directly, serving as the control. To further improve protein identification, we also revised mass spectrometer parameters such as normalized collision energy, ion trap time, and dynamic exclusion time for MS2. This systematic approach helped increase the number of protein identifications from ~600 to ~900 protein groups in the control sample. The high pH fractionation led to identification of more than 1,400 protein groups, corresponding to ~35% increase in identifications compared to the control. The additional ~500 proteins identified by high pH fractionation corresponded to medium- to low-abundance proteins. At present, we are developing this approach to further improve protein identifications. We aim to use the method to quantify protein regulation as the embryo undergoes successive stages of early development to gain a deeper understanding of gene translation during vertebrate embryonic development.
Creative Commons License
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Open Access
1
High pH Fractionation for Enhancing the Proteome Coverage in Tissues Dissected from the Early Frog (Xenopus laevis) Embryo
Discovery measurement of the proteome raises an opportunity to better understand how differential expression of the genome underlies the formation of various tissue types during early embryonic development. To enhance the detectable proteome, we and others have developed high-sensitivity mass spectrometry instruments and protocols for the removal of abundant proteins, such as pelleting of yolk proteins using density gradients in the frog (Xenopus laevis) embryo. Here, we test high pH fractionation to improve the identification and quantification of proteins in the early developing X. laevis embryo.
The main goal of this work was to enhance the identification and quantification of proteins in the early developing frog (Xenopus laevis) embryo. While X. laevis is a powerful model of developmental biology and health studies, the proteome of the early embryo is dominated by abundant yolk proteins (>90% of the proteome), which hinders the characterization of low-abundance proteins. Building on the success of high pH fractionation, we proposed that this approach would help improve protein identifications in Xenopus tissues by minimizing the complexity of the samples prior to liquid chromatography-Mass spectrometry analysis. To test this, we pooled tissues from n = 10 embryos and extracted proteins using a SDS based lysis buffer and precipitated the proteins overnight in cold acetone. The extracted proteins were then digested with trypsin for 5 h at 37 ºC. The resulting peptides were split into two sections: one was processed further by high-pH fractionation and the other was measured directly, serving as the control. To further improve protein identification, we also revised mass spectrometer parameters such as normalized collision energy, ion trap time, and dynamic exclusion time for MS2. This systematic approach helped increase the number of protein identifications from ~600 to ~900 protein groups in the control sample. The high pH fractionation led to identification of more than 1,400 protein groups, corresponding to ~35% increase in identifications compared to the control. The additional ~500 proteins identified by high pH fractionation corresponded to medium- to low-abundance proteins. At present, we are developing this approach to further improve protein identifications. We aim to use the method to quantify protein regulation as the embryo undergoes successive stages of early development to gain a deeper understanding of gene translation during vertebrate embryonic development.
Comments
To be presented at GW Annual Research Days 2017.