The insoluble fraction obtained after 0.2% TFA addition (1:1, v/v) to saliva of preterm human newborns was analyzed by 2D-PAGE in order to evaluate its protein composition with respect to the soluble fraction, already extensively characterized by ESI-mass spectrometry [1]. A high number of proteins has been identified by high-resolution HPLC−ESI−MS/MS experiments performed after in-gel trypsin digestion of the spots by an LTQ Orbitrap XL apparatus. Some of them have never been found in the soluble fraction, while some others appeared present both in the soluble and insoluble fractions (i.e. S100A8, S100A9, S100A11 and cystatin-B). S100A8 has been identified in three spots, two aligned at a MW < 13 kDa and over a 6.0-7.0 pH range, the third, localized at a very high MW (>100 kDa), and aligned with the highest S100A8 pI. According to the paper of Lim et al. [2] we hypothesized that it may represent a DTT-resistant covalent complex of oxidized S100A8 molecules. Interestingly, several spots on the 2D-PAGE appeared as trains of spots, that may be related to phosphorylation, sulfation or other PTMs, that we are going to evaluate, after electroelution of each spot, by high-resolution HPLC−ESI−MS/MS analysis. The future aim of this study is the analysis of the insoluble fraction of the salivary proteome of preterm newborns as a function of postconceptional age (PCA) to verify if the concentration decreasing trend as PCA increases, observed for the 25 proteins identified with high frequency in the soluble fraction, is present also in the proteins identified in the insoluble fraction.
Proteomic analysis of the insoluble fraction of preterm human newborn saliva
ARBA, MORENA;MANCONI, BARBARA;OLIANAS, ALESSANDRA;SANNA, MARIA TERESA
2013-01-01
Abstract
The insoluble fraction obtained after 0.2% TFA addition (1:1, v/v) to saliva of preterm human newborns was analyzed by 2D-PAGE in order to evaluate its protein composition with respect to the soluble fraction, already extensively characterized by ESI-mass spectrometry [1]. A high number of proteins has been identified by high-resolution HPLC−ESI−MS/MS experiments performed after in-gel trypsin digestion of the spots by an LTQ Orbitrap XL apparatus. Some of them have never been found in the soluble fraction, while some others appeared present both in the soluble and insoluble fractions (i.e. S100A8, S100A9, S100A11 and cystatin-B). S100A8 has been identified in three spots, two aligned at a MW < 13 kDa and over a 6.0-7.0 pH range, the third, localized at a very high MW (>100 kDa), and aligned with the highest S100A8 pI. According to the paper of Lim et al. [2] we hypothesized that it may represent a DTT-resistant covalent complex of oxidized S100A8 molecules. Interestingly, several spots on the 2D-PAGE appeared as trains of spots, that may be related to phosphorylation, sulfation or other PTMs, that we are going to evaluate, after electroelution of each spot, by high-resolution HPLC−ESI−MS/MS analysis. The future aim of this study is the analysis of the insoluble fraction of the salivary proteome of preterm newborns as a function of postconceptional age (PCA) to verify if the concentration decreasing trend as PCA increases, observed for the 25 proteins identified with high frequency in the soluble fraction, is present also in the proteins identified in the insoluble fraction.
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