The potential of proteomics for in-depth bioanalytical investigations of satellite cell function in applied myology

Figures & data

Figure 1. Overview of the involvement of muscle stem cells in regenerative myogenesis. The color of the transcription factors marks their presence (green) versus absence (red) in specific cell types. FAPs, fibro-adipogenic progenitors; MRFs, myogenic regulatory factors; MSCs, mesenchymal stem cells/multipotent stromal cells; MYOD1, myoblast determination protein 1; MyHC, myosin heavy chain; MYOG, muscle-specific transcription factor myogenin; PAX7, paired box 7; SCs, satellite cells.

Figure 2. Bioinformatic STRING analysis of major myosin components in the sarcomere of mouse skeletal muscles. MyHC, myosin heavy chain; MLC, myosin light chain; MYBP, myosin-binding protein.

Table 1. Listing of the main cell types that are involved in embryonic myogenesis versus adult regenerative myogenesis, and their markers.

Cell types	Markers
(i) Embryonic myogenesis
Progenitor/stem cells	PAX3, PAX7
1st progenitor	MYF5, MRF4, MYOD1
2nd progenitor	MYF5, MYOD1
Embryonic/fetal myoblasts	MYF5, MYOD1
1st/2nd embryonic myofiber	MYOG
(ii) Adult regenerative myogenesis
Quiescent satellite cells	PAX7, FOXO
Activated satellite cells	PAX7, MYF5, MYOD1
Myoblasts	PAX7±, MYF5, MYOD1
Myocytes	MYOD1, MYOG, MRF4
Myofiber	MYOG, MRF4, MyHC

*Abbreviations used: FOXO, forkhead box O; MRF4, myogenic regulatory factor 4; MYF5, myogenic factor 5; MyHC, myosin heavy chain; MYOD1, myoblast determination protein 1; MYOG, myogenin; PAX, paired box.

Figure 3. Proteomic profiling strategy to study skeletal muscles with a special focus on the muscle stem cell niche. Shown is the analytical workflow to study total muscle protein extracts from tissue sources, muscle-derived cell types following culturing and single-cell analysis with the help of flow cytometric sorting and separation. Frequently used methods for mass spectrometry-based protein identification and data acquisition, muscle cell analysis using stable isotope labeling and single-cell proteomics are listed. 2D-GE, two-dimensional gel electrophoresis; DDA, data-dependent acquisition; DIA, data-independent acquisition; ESI, electrospray ionization; FACS, fluorescence-activated cell sorting; FT, Fourier-transform ion cyclotron resonance; GeLC, gel electrophoresis-liquid chromatography; iBASIL, Improved Boosting to Amplify Signal with Isobaric Labeling; ICAT, isotope-coded affinity tags; ICPL, isotope-coded protein labeling; iTRAQ, isobaric tagging for relative and absolute quantitation; LC, liquid chromatography; LFQ, label-free quantification; MALDI-ToF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; MudPIT; multi-dimensional protein identification technology; PRM, parallel reaction monitoring; PTMs, post-translational modifications; SCoPE, Single Cell ProtEomics by Mass Spectrometry; SILAC, stable isotope labeling by amino acids in cell culture; SRM/MRM, selected/multiple reaction monitoring; SWATH, Sequential Window Acquisition of all Theoretical Mass Spectra; TDA, targeted data acquisition; TMT, tandem mass tags.

Table 2. Overview of major proteomic approaches and their bioanalytical advantages versus technical limitations.

Proteomic methodology	Advantages	Disadvantages/limitations
Gel-based and top-down proteomics
Two-dimensional gel electrophoresis (2D-GE) coupled with mass spectrometry (MS)	Individual 2D protein spots displaying unique proteoforms are highly suitable for MS-based protein identification	High-/low-molecular weight proteins and hydrophobic proteins are difficult to separate and analyze; 2D-GE has low throughput
Fluorescence two-dimensional difference in-gel electrophoresis (2D-DIGE) coupled with MS analysis	Multiplexing of samples; greatly reduced gel-to-gel variations; highly sensitive fluorescent tagging of proteins of interest	Using CyDyes for differential tagging adds a significant cost to comparative proteomic experiments
Gel-free and bottom-up proteomics
Stable isotope labeling by amino acids in cell culture (SILAC)	Multiplexing (pairwise comparisons); compatibility with complex fractionation procedures	Mostly limited to tissue culture models; not applicable to tissue/biofluid samples
Tandem mass tags (TMT)-based analysis	Multiplexing is compatible with any sample type; accurate quantitation	Co-eluting peptides with similar molecular weights can affect quantitation of reporter ions; variable efficiency in labeling reported
Label-free quantification (LFQ) analysis	LFQ approach avoids the usage of expensive labels; streamlined protocols	Multiplexed analysis is not possible in a single experiment; lower accuracy of quantification compared to labeling methods; high technical and experimental reproducibility needed (retention times)
Single-cell proteomics (SCP)	SCP allows cellular heterogeneity to be uncovered at single-cell resolution; enables comparison of SCP data to other single-cell platforms (transcriptomics)	High levels of technical challenges encountered; dedicated microfluidics and MS instrumentations necessary