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Characterizing Qualitative and Quantitative Global Changes in the Aging Heart Using pSMART, a Novel Acquisition Method
Overview
Purpose:
We have completed a label-free quantitative global profiling and targeted
analysis of the cardiac proteome in aging mice using a novel data acquisition method,
pSMART.
Methods:
The pSMART method was used to acquire qualitative and quantitative data
using one HR/AM MS and a series of narrow DIA mass windows. Initial
characterization experiments using unbiased DDA facilitated the building of a detailed
spectral library which was used for qualitative scoring for the DIA data.
Results:
Our pSMART strategy resulted in 30% more peptide identifications per run
than a standard DDA run. Additionally, using pSMART, we are able to confirm MS1
quantitation at low abundance levels with MS/MS for each peptide. This novel
acquisition enabled quantitation of previously identified peptides as well as novel
putative targets of aging. By identifying and quantifying more targets, we were able to
better characterize the dynamic proteomic changes of cardio-dysfunction in aging
mice.
Introduction
The cardiovascular system undergoes significant changes as it ages. Aging is a
complex event that eventually leads to loss of function and abilities over time and has
been shown to cause multi-level changes in the heart, from the genomic-transcriptomic
level to the cellular-tissue level. These pathological changes in the heart can be
followed at the proteomic level.
In this study we introduce a complete workflow that enables label-free quantification of
age-related proteomic changes in murine heart tissue.
Methods
Sample Preparation
Heart tissue was isolated and homogenized from young (2 months old) and old (2
years old) C57BL/6 mice. Each heart was normalized by weight, homogenized in 8M
GuHCl, 250 mM Tris, pH 8.5, using a FastPrep 120. The supernatant was then
reduced, alkylated and digested with sequencing grade Thermo Scientific
TM
Pierce
TM
trypsin kit overnight. A portion of each of the digested samples was then separated by
high pH reverse-phase fractionation into 16 equal fractions. These fractions were used
to create the spectral library. Each sample was spiked with Peptide Retention Time
Calibration Mixture (PRTC) (Thermo Fisher Scientific Pierce) prior to mass
spectrometry analysis.
Mass Spectrometry and Data Analysis
All samples were analyzed on a Thermo Scientific™
Orbitrap Fusion
™
Tribrid
TM
mass
spectrometer equipped with a Thermo Scientific™
Nanospray Flex
TM
Ion Source. Data
were acquired in two steps to simulate traditional workflows. Initial experiments
employed unbiased data-dependent MS/MS acquisition (DDA) for each of the
fractionated samples to build the spectral library. Each non-fractionated heart sample
was also run with unbiased DDA as well as with our novel pSMART data acquisition
method (Figure 1). Each non-fractionated heart sample was run in triplicate.
Thermo Scientific™ Proteome Discoverer™ version 1.4 and Thermo Scientific™
Pinpoint™ version 1.4 software packages were used to analyze both the qualitative
and quantitative data. The spectral library resulting from initial fractionated sample runs
was used to create reference information to perform confirmation on all peptide targets.
FIGURE1. pSMART data ac
mass (HR/AM) MS and 5 Da
consists of two independen
acquisition in between eac
quantitative (fullscan MS) a
most sensitive global quant
acquisition cycle times ass
precursor m/z values under
Results
FIGURE2. Biomarker discov
step process consisting of
initial, unbiased characteriza
to create the spectral library
Global quantitation is perfor
Pinpoint. The list of peptide
product ion information are r
processing.