Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy.

Mass spectrometry (MS)-based top-down proteomics is a powerful method for the comprehensive analysis of proteoforms that arise from genetic variations and post-translational modifications (PTMs). However, top-down MS analysis of high molecular weight (MW) proteins remains challenging mainly due to the exponential decay of signal-to-noise ratio with increasing MW. Size exclusion chromatography (SEC) is a favored method for size-based separation of biomacromolecules but typically suffers from low resolution. Herein, we developed a serial size exclusion chromatography (sSEC) strategy to enable high-resolution size-based fractionation of intact proteins (10-223 kDa) from complex protein mixtures. The sSEC fractions could be further separated by reverse phase chromatography (RPC) coupled online with high-resolution MS. We have shown that two-dimensional (2D) sSEC-RPC allowed for the identification of 4044 more unique proteoforms and a 15-fold increase in the detection of proteins above 60 kDa, compared to one-dimensional (1D) RPC. Notably, effective sSEC-RPC separation of proteins significantly enhanced the detection of high MW proteins up to 223 kDa and also revealed low abundance proteoforms that are post-translationally modified. This sSEC method is MS-friendly, robust, and reproducible and, thus, can be applied to both high-efficiency protein purification and large-scale proteomics analysis of cell or tissue lysate for enhanced proteome coverage, particularly for low abundance and high MW proteoforms.

The function of platelet-derived growth factor in the differentiation of mouse tongue striated muscle.

OBJECTIVE: To determine the function of platelet-derived growth factor (PDGF) in the final differentiation phase of tongue striated muscle cells. MATERIALS AND METHODS: We analyzed the expressions of PDGF-A, -B, platelet-derived growth factor receptor (PDGFR)-α, and PDGFR-ß in mouse tongues between embryonic days (E) 11 and 15. Furthermore, we examined the effects of human recombinant PDGF-AB and the peptide antagonist for PDGFRs using an organ culture system of mouse embryonic tongue. Mouse tongues at E12 were cultured in BGJb medium containing human recombinant PDGF-AB for 4 days or the peptide antagonist for PDGF receptors for 8 days. RESULTS: PDGF-A, -B, PDGFR-α, and -ß were expressed in the differentiating muscle cells between E11 and 15. The human recombinant PDGF-AB induced increases in the mRNA expressions of myogenin and muscle creatine kinase (MCK) and the number of fast myosin heavy chain (fMHC)-positive cells, markers for the differentiation of muscle cells. On the other hand, the peptide antagonist for PDGFRs induced suppressions in the mRNA expressions of myogenin and MCK, and the number of fMHC-positive cells. Both the PDGF-AB and the antagonist failed to affect the expressions of cell proliferation markers. CONCLUSION: These results suggest that PDGF functions as a positive regulator in the final differentiation phase of tongue muscle cells in mouse embryos.

Progressive decrease of phosphocreatine, creatine and creatine kinase in skeletal muscle upon transformation to sarcoma.

In vertebrates, phosphocreatine and ATP are continuously interconverted by the reversible reaction of creatine kinase in accordance with cellular energy needs. Sarcoma tissue and its normal counterpart, creatine-rich skeletal muscle, are good source materials to study the status of creatine and creatine kinase with the progression of malignancy. We experimentally induced sarcoma in mouse leg muscle by injecting either 3-methylcholanthrene or live sarcoma 180 cells into one hind leg. Creatine, phosphocreatine and creatine kinase isoform levels decreased as malignancy progressed and reached very low levels in the final stage of sarcoma development; all these parameters remained unaltered in the unaffected contralateral leg muscle of the same animal. Creatine and creatine kinase levels were also reduced significantly in frank malignant portions of human sarcoma and gastric and colonic adenocarcinoma compared with the distal nonmalignant portions of the same samples. In mice, immunoblotting with antibodies against cytosolic muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase showed that both of these isoforms decreased as malignancy progressed. Expressions of mRNA of muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase were also severely downregulated. In human sarcoma these two isoforms were undetectable also. In human gastric and colonic adenocarcinoma, brain-type creatine kinase was found to be downregulated, whereas ubiquitous mitochondrial creatine kinase was upregulated. These significantly decreased levels of creatine and creatine kinase isoforms in sarcoma suggest that: (a) the genuine muscle phenotype is lost during sarcoma progression, and (b) these parameters may be used as diagnostic marker and prognostic indicator of malignancy in this tissue.

Significance of CK-elevation in noncompaction with regard to cardiac and neuromuscular disease.

OBJECTIVES: This retrospective study aimed to find out how often CK is elevated in left-ventricular hypertrabeculation/noncompaction (LVHT), how often CK-elevation is due to neuromuscular disorders (NMDs), other cardiac disease, or causes other than cardiac or NMD, and how often CK-elevation is associated with troponin-T-positivity. METHODS: Electronic records from all LVHT-patients diagnosed between 1995-2005 were reviewed for CK, CK-MB, and troponin-T-values, frequency of CK-elevation, and causes of CK-elevation. RESULTS: Among 100 LVHT-patients, electronic records with laboratory data and final reports were available in 88. The highest CK-values ranged between 9-5547U/l. In 40 patients at least one CK-value was elevated (46%). Only 8 patients were troponin-T-positive. There was no significant difference of the clinical cardiologic, ECG, and echocardiographic parameters between patients with normal and elevated CK. Seventy of the 88 patients were seen by a neurologist. A NMD was diagnosed in 57 patients (81%). CK-elevation was attributed to NMD in 31 patients (79%), to causes other than cardiac or NMD in 6 patients (15%), and to cardiac causes in 4 patients (10%). CONCLUSIONS: CK-elevation occurs in half of the LVHT-patients. CK-elevation in LVHT is most frequently attributable to NMDs but hardly to cardiac disease. CK-elevation in LVHT suggests NMD and prompts neurological investigations.

Detection of local polarity and conformational changes at the active site of rabbit muscle creatine kinase with a new arginine-specific fluorescent probe.

A new polarity-sensitive fluorescent probe, 3-(4-chloro-6-p-glyoxal-phenoxy-1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine (CGTDP), is synthesized for selective labeling of active-site arginine residues. The probe comprises a neutral red moiety as a polarity-sensitive fluorophore and a phenylglyoxal unit as an arginine-specific labeling group. The probe exhibits a sensitive response of shift of fluorescence maximum emission wavelength to solvent polarity only instead of pH or temperature, which leads to the use of the probe in detecting the local polarity and conformational changes of the active site of rabbit muscle creatine kinase (CK) denatured by pH or temperature. The polarity of the active site domain has been first found to correspond to a dielectric constant of about 44, and the conformational change of the active site directly revealed by CGTDP occurs far before that of CK as a whole disclosed by the intrinsic tryptophan fluorescence during acid or thermal denaturation. The present strategy may provide a useful method to detect the local polarity and conformational changes of the active sites of many enzymes that employ arginine residues as anion recognition sites under different denaturation conditions.

Finasteride-induced myalgia and HyperCKemia.

BACKGROUND: Finasteride is an antiandrogen, inhibits type II 5-alpha reductase (enzyme that converts testosterone to more potent form dihydrotestosterone), and is commonly used in the treatment of benign prostatic hyperplasia and male frontal baldness; however, it is not free from side effects, which include sexual dysfunction and, rarely, myopathy. We report a case of finasteride-associated myalgia and hyperCKemia and review similar cases reported in the literature. CASE REPORT: A 30-year-old man who had been taking finasteride 5 mg/d for 10 years to treat frontal baldness developed diffuse muscle aches associated with elevated creatine kinase level to 10,117 IU/L with neither weakness nor pigmenturia. His symptoms resolved and his creatine kinase level dropped down to 256 IU/L 3 weeks after finasteride discontinuation. CONCLUSION: Reversible myalgia associated with significant hyperCKemia is a possible adverse reaction of finasteride therapy.

Proteomic analysis of organ-specific post-translational lysine-acetylation and -methylation in mice by use of anti-acetyllysine and -methyllysine mouse monoclonal antibodies.

Post-translational lysine-acetylation and -methylation are two major PTMs of lysine residues in proteins. Recently, we established pan-reactive anti-acetyllysine mouse mAbs, which can bind to Nepsilon-acetylated lysine residues in various contexts of amino acid sequences. In the present study, we established pan-reactive anti-methyllysine mouse mAbs comparable to the anti-acetyllysine ones. By using these anti-acetyllysine and -methyllysine antibodies, we found that the pattern of lysine-acetylated and -methylated proteins in mouse organs showed extreme variation from organ to organ. We selected brain and skeletal muscle as model cases to be further analyzed by 2-DE followed by Western blotting. In brain, alpha-tubulin at its basal level was found to be extremely acetylated; and alpha-enolase was shown to be a newly recognized possibly acetylated protein. NF-L protein, Hsc70, alpha-tubulin fragments, beta-actin, and brain-type creatine kinase were identified as putative lysine-methylated proteins in mouse brain. In skeletal muscle, lysine-methylation of alpha-actin and both lysine-acetylation and -methylation of muscle-type creatine kinase were found as novel putative lysine-modified proteins. The approach presented here might be useful to find novel disease markers and/or drug target molecules that would not be noticed by use of the traditional proteomic approach only.