Universal proteomics sample preparation is challenging because of the high heterogeneity of biological samples. Here we describe a novel mechanism that exploits the inherent instability of denatured proteins for nonspecific immobilization on microparticles by protein aggregation capture. To demonstrate the general applicability of this mechanism, we analyzed phosphoproteomes, tissue proteomes, and interaction proteomes as well as dilute secretomes. The findings present a practical, sensitive and cost-effective proteomics sample preparation method.

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.

AMP-activated protein kinase (AMPK) is a heterotrimeric protein that regulates glucose transport mediated by cellular stress or pharmacological agonists such as 5-aminoimidazole-4-carboxamide 1 β-d-ribonucleoside (AICAR). AS160, a Rab GTPase-activating protein, provides a mechanism linking AMPK signaling to glucose uptake. We show that AICAR increases AMPK, acetyl-CoA carboxylase, and AS160 phosphorylation by insulin-independent mechanisms in isolated skeletal muscle. Recombinant AMPK heterotrimeric complexes (α1β1γ1 and α2β2γ1) phosphorylate AS160 in a cell-free assay. In mice deficient in AMPK signaling (α2 AMPK knockout [KO], α2 AMPK kinase dead [KD], and γ3 AMPK KO), AICAR effects on AS160 phosphorylation were severely blunted, highlighting that complexes containing α2 and γ3 are necessary for AICAR-stimulated AS160 phosphorylation in intact skeletal muscle. Contraction-mediated AS160 phosphorylation was also impaired in α2 AMPK KO and KD but not γ3 AMPK KO mice. Our results implicate AS160 as a downstream target of AMPK.

Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high‐sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype‐specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type‐resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity. Synopsis Here, single multinucleated muscle fibers are analyzed by high sensitivity proteomics for the first time. This reveals an unexpected diversity and specialization in metabolic properties of individual fiber types, in particular in mitochondrial function. First single skeletal muscle fiber proteome, and thereby single cell proteome of any kind Single‐shot proteomic analysis captures main features of type 1‐slow and of the subtypes of fast fibers Qualitative differences in the mitochondrial proteome of the four fiber types uncover a metabolic specialization