5 research outputs found
A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics
Skeletal muscle is a major regulatory tissue of whole-body
metabolism
and is composed of a diverse mixture of cell (fiber) types. Aging
and several diseases differentially affect the various fiber types,
and therefore, investigating the changes in the proteome in a fiber-type
specific manner is essential. Recent breakthroughs in isolated single
muscle fiber proteomics have started to reveal heterogeneity among
fibers. However, existing procedures are slow and laborious, requiring
2 h of mass spectrometry time per single muscle fiber; 50 fibers would
take approximately 4 days to analyze. Thus, to capture the high variability
in fibers both within and between individuals requires advancements
in high throughput single muscle fiber proteomics. Here we use a single
cell proteomics method to enable quantification of single muscle fiber
proteomes in 15 min total instrument time. As proof of concept, we
present data from 53 isolated skeletal muscle fibers obtained from
two healthy individuals analyzed in 13.25 h. Adapting single cell
data analysis techniques to integrate the data, we can reliably separate
type 1 and 2A fibers. Ninety-four proteins were statistically different
between clusters indicating alteration of proteins involved in fatty
acid oxidation, oxidative phosphorylation, and muscle structure and
contractile function. Our results indicate that this method is significantly
faster than prior single fiber methods in both data collection and
sample preparation while maintaining sufficient proteome depth. We
anticipate this assay will enable future studies of single muscle
fibers across hundreds of individuals, which has not been possible
previously due to limitations in throughput
A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics
Skeletal muscle is a major regulatory tissue of whole-body
metabolism
and is composed of a diverse mixture of cell (fiber) types. Aging
and several diseases differentially affect the various fiber types,
and therefore, investigating the changes in the proteome in a fiber-type
specific manner is essential. Recent breakthroughs in isolated single
muscle fiber proteomics have started to reveal heterogeneity among
fibers. However, existing procedures are slow and laborious, requiring
2 h of mass spectrometry time per single muscle fiber; 50 fibers would
take approximately 4 days to analyze. Thus, to capture the high variability
in fibers both within and between individuals requires advancements
in high throughput single muscle fiber proteomics. Here we use a single
cell proteomics method to enable quantification of single muscle fiber
proteomes in 15 min total instrument time. As proof of concept, we
present data from 53 isolated skeletal muscle fibers obtained from
two healthy individuals analyzed in 13.25 h. Adapting single cell
data analysis techniques to integrate the data, we can reliably separate
type 1 and 2A fibers. Ninety-four proteins were statistically different
between clusters indicating alteration of proteins involved in fatty
acid oxidation, oxidative phosphorylation, and muscle structure and
contractile function. Our results indicate that this method is significantly
faster than prior single fiber methods in both data collection and
sample preparation while maintaining sufficient proteome depth. We
anticipate this assay will enable future studies of single muscle
fibers across hundreds of individuals, which has not been possible
previously due to limitations in throughput
A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics
Skeletal muscle is a major regulatory tissue of whole-body
metabolism
and is composed of a diverse mixture of cell (fiber) types. Aging
and several diseases differentially affect the various fiber types,
and therefore, investigating the changes in the proteome in a fiber-type
specific manner is essential. Recent breakthroughs in isolated single
muscle fiber proteomics have started to reveal heterogeneity among
fibers. However, existing procedures are slow and laborious, requiring
2 h of mass spectrometry time per single muscle fiber; 50 fibers would
take approximately 4 days to analyze. Thus, to capture the high variability
in fibers both within and between individuals requires advancements
in high throughput single muscle fiber proteomics. Here we use a single
cell proteomics method to enable quantification of single muscle fiber
proteomes in 15 min total instrument time. As proof of concept, we
present data from 53 isolated skeletal muscle fibers obtained from
two healthy individuals analyzed in 13.25 h. Adapting single cell
data analysis techniques to integrate the data, we can reliably separate
type 1 and 2A fibers. Ninety-four proteins were statistically different
between clusters indicating alteration of proteins involved in fatty
acid oxidation, oxidative phosphorylation, and muscle structure and
contractile function. Our results indicate that this method is significantly
faster than prior single fiber methods in both data collection and
sample preparation while maintaining sufficient proteome depth. We
anticipate this assay will enable future studies of single muscle
fibers across hundreds of individuals, which has not been possible
previously due to limitations in throughput
A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics
Skeletal muscle is a major regulatory tissue of whole-body
metabolism
and is composed of a diverse mixture of cell (fiber) types. Aging
and several diseases differentially affect the various fiber types,
and therefore, investigating the changes in the proteome in a fiber-type
specific manner is essential. Recent breakthroughs in isolated single
muscle fiber proteomics have started to reveal heterogeneity among
fibers. However, existing procedures are slow and laborious, requiring
2 h of mass spectrometry time per single muscle fiber; 50 fibers would
take approximately 4 days to analyze. Thus, to capture the high variability
in fibers both within and between individuals requires advancements
in high throughput single muscle fiber proteomics. Here we use a single
cell proteomics method to enable quantification of single muscle fiber
proteomes in 15 min total instrument time. As proof of concept, we
present data from 53 isolated skeletal muscle fibers obtained from
two healthy individuals analyzed in 13.25 h. Adapting single cell
data analysis techniques to integrate the data, we can reliably separate
type 1 and 2A fibers. Ninety-four proteins were statistically different
between clusters indicating alteration of proteins involved in fatty
acid oxidation, oxidative phosphorylation, and muscle structure and
contractile function. Our results indicate that this method is significantly
faster than prior single fiber methods in both data collection and
sample preparation while maintaining sufficient proteome depth. We
anticipate this assay will enable future studies of single muscle
fibers across hundreds of individuals, which has not been possible
previously due to limitations in throughput
A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics
Skeletal muscle is a major regulatory tissue of whole-body
metabolism
and is composed of a diverse mixture of cell (fiber) types. Aging
and several diseases differentially affect the various fiber types,
and therefore, investigating the changes in the proteome in a fiber-type
specific manner is essential. Recent breakthroughs in isolated single
muscle fiber proteomics have started to reveal heterogeneity among
fibers. However, existing procedures are slow and laborious, requiring
2 h of mass spectrometry time per single muscle fiber; 50 fibers would
take approximately 4 days to analyze. Thus, to capture the high variability
in fibers both within and between individuals requires advancements
in high throughput single muscle fiber proteomics. Here we use a single
cell proteomics method to enable quantification of single muscle fiber
proteomes in 15 min total instrument time. As proof of concept, we
present data from 53 isolated skeletal muscle fibers obtained from
two healthy individuals analyzed in 13.25 h. Adapting single cell
data analysis techniques to integrate the data, we can reliably separate
type 1 and 2A fibers. Ninety-four proteins were statistically different
between clusters indicating alteration of proteins involved in fatty
acid oxidation, oxidative phosphorylation, and muscle structure and
contractile function. Our results indicate that this method is significantly
faster than prior single fiber methods in both data collection and
sample preparation while maintaining sufficient proteome depth. We
anticipate this assay will enable future studies of single muscle
fibers across hundreds of individuals, which has not been possible
previously due to limitations in throughput