Quantitative temporal analysis of protein dynamics in cardiac remodeling

Abstract

Cardiac remodeling (CR) is a complex dynamic process common to many heart diseases. CR is characterized as a temporal progression of global adaptive and maladaptive perturbations. The complex nature of this process clouds a comprehensive understanding of CR, but greater insight into the processes and mechanisms has potential to identify new therapeutic targets. To provide a deeper understanding of this important cardiac process, we applied a new proteomic technique, PALM (Pulse Azidohomoalanine in Mammals), to quantitate the newly-synthesized protein (NSP) changes during the progression of isoproterenol (ISO)-induced CR in the mouse left ventricle. This analysis revealed a complex combination of adaptive and maladaptive alterations at acute and prolonged time points including the identification of proteins not previously associated with CR. We also combined the PALM dataset with our published protein turnover rate dataset to identify putative biochemical mechanisms underlying CR. The novel integration of analyzing NSPs together with their protein turnover rates demonstrated that alterations in specific biological pathways (e.g., inflammation and oxidative stress) are produced by differential regulation of protein synthesis and degradation.

Quantitative analysis of global protein stability rates in tissues

“…capillary with a 5 μm pulled tip packed with 15 cm 4 μm Jupiter C18 material (Phenomenex, Ventura, CA) was attached to the loading column with a union, and the entire split-column(loading column-union-analytical column) was placed in-line with an Agilent 1,100 quaternary HPLC (Palo Alto, CA). The sample was analyzed using an eleven step MudPIT, which is a salt-step separation previously described 22 . As peptides eluted from the microcapillary column, they were electrosprayed directly into a Velos mass spectrometer (ThermoFisher, Palo Alto, CA) with the application of a distal 2.4 kV spray voltage.…”

“…For the analysis described in Fig. 4 E,F, a nano-Easy HPLC (ThermoFisher) with an Elite mass spectrometer (ThermoFisher) was used with the MS settings previously described 22 .…”

“…In fact, AHA has been employed as an infrared spectroscopy probe to study native protein structure and folding 19 – 21 . Numerous biological studies have used AHA to study the most delicate and fragile proteomes with no disruption of function 22 – 24 . Most relevant to this study, AHA labeled proteins have been shown to be as stable as native proteins in cultured cells 25 , 26 .…”

Quantitative Analysis of Global Protein Stability Rates in Tissues

“…capillary with a 5 μm pulled tip packed with 15 cm 4 μm Jupiter C18 material (Phenomenex, Ventura, CA) was attached to the loading column with a union, and the entire split-column(loading column−union−analytical column) was placed inline with an Agilent 1100 quaternary HPLC (Palo Alto, CA). The sample was analyzed using MudPIT, which is an eleven salt-step separation previously described [75]. As peptides eluted from the microcapillary column, they were electrosprayed directly into a Velos mass spectrometer (ThermoFisher, Palo Alto, CA) with the application of a distal 2.4 kV spray voltage.…”

“…A cycle of one full-scan FT mass spectrum (300−1600 m/z) at 60 000 resolution followed by 20 data-dependent IT MS/MS spectra at a 35% normalized collision energy was repeated continuously throughout each step of the multidimensional separation. For the analysis in Figure 8, a nano-Easy HPLC (ThermoFisher) with an Elite mass spectrometer (ThermoFisher) was used with the MS setting previously described [75].…”