Phosphoproteome profile of human liver Chang's cell based on 2‐DE with fluorescence staining and MALDI‐TOF/TOF‐MS

Liu, Jinfeng; Cai, Yun; Wang, Jinglan; Zhou, Qi; Yang, Bing; Lu, Zhenwu; Jiao, Liyan; Zhang, Dongyang; Sui, Shaohui; Jiang, Ying; Ying, Wantao; Qian, Xiaohong

doi:10.1002/elps.200600696

Cited by 18 publications

(10 citation statements)

References 28 publications

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“…Prior to the comparative gel electrophoretic analysis of the young adult versus aged skeletal muscle phosphoproteome, we determined the position of landmark proteins with a phosphorylation moiety. This analysis was carried out with the fluorescent phosphosensitive ProQ Diamond labeling method (48,49), which has recently been applied in several large-scale phosphoproteomic studies (53)(54)(55). Fig.…”

Section: Resultsmentioning

confidence: 99%

Phosphoproteomic analysis of aged skeletal muscle

Gannon

Staunton²,

O’Connell³

et al. 2008

Int J Mol Med

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Abstract. One of the most important post-translational modifications is represented by phosphorylation on tyrosine, threonine and serine residues. Since abnormal phosphorylation is associated with various pathologies, it was of interest to perform a phosphoproteomic profiling of age-related skeletal muscle degeneration. We used the fluorescent phosphospecific Pro-Q Diamond dye to determine whether changes in the overall phosphorylation of the soluble skeletal muscle proteome differs significantly between young adult and senescent fibres. As an established model system of sarcopenia, we employed 30-month-old rat gastrocnemius fibres. Following the mass spectrometric identification of 59 major 2-D phosphoprotein landmark spots, the fluorescent dye staining survey revealed that 22 muscle proteins showed a differential expression pattern between 3-month-and 30-month-old muscle. Increased phosphorylation levels were shown for myosin light chain 2, tropomyosin α, lactate dehydrogenase, desmin, actin, albumin and aconitase. In contrast, decreased phospho-specific dye binding was observed for cytochrome c oxidase, creatine kinase and enolase. Thus, aging-induced alterations in phosphoproteins appear to involve the contractile machinery and the cytoskeleton, as well as the cytosolic and mitochondrial metabolism. This confirms that sarcopenia of old age is a complex neuromuscular pathology that is associated with drastic changes in the abundance and structure of key muscle proteins.

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Section: Resultsmentioning

confidence: 99%

Phosphoproteomic analysis of aged skeletal muscle

Gannon

Staunton²,

O’Connell³

et al. 2008

Int J Mol Med

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“…2-D gel with Coomassie G250 staining is a widely used technique for quantitative protein analysis [17,[26][27][28].…”

Section: Discussionmentioning

confidence: 99%

Quantitative proteomic analysis of lentiviral vectors using 2‐DE

et al. 2009

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Among the integrative gene therapy vectors developed to date, human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LV) are distinguished by their capacity to infect both dividing and non-dividing cells. Recombinant LV particles contain viral proteins necessary for their packaging, infectious and integrating functions. Like the parental HIV-1 virus they are able to acquire various cellular proteins, but the number and localisation of these proteins are poorly characterised. In the present study we used 2-DE followed by MALDI-TOF to quantify the protein content of several types of vesicular stomatitis virus G-pseudotyped LV including those that were extensively purified in the perspective of clinical gene therapy studies. A proteinase K treatment was used to distinguish between cellular proteins incorporated into virions (I-proteins) and those co-purified with vectors (C-proteins). We found 10 C-proteins and 18 I-proteins associated with LV. Copy numbers for these core I-proteins varied from 5 (AIP-1/ALIX) to 280 (Cyclophilin A) per vector particle. Three novel I-proteins, guanine nucleotide-binding protein 2, L-lactate dehydrogenase B chain and hnRNP core protein A1, were found. This study defines for the first time, the protein stoichiometry of infectious HIV-1-derived LV particles.

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“…Following the image acquisition, gels were stained with Sypro ruby protein stain (Molecular Probes) to detect total protein. The serial dichromatic detections were used to compare relative levels of phosphorylation of a given protein [20, 21]. …”

Section: Methodsmentioning

confidence: 99%

Proteomic profiling of phosphoproteins and glycoproteins responsive to wild-type alpha-synuclein accumulation and aggregation

Kulathingal

Cusack

et al. 2009

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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A tetracycline inducible transfectant cell line (3D5) capable of producing soluble and sarkosylinsoluble assemblies of wild-type human alpha-synuclein (α-Syn) upon differentiation with retinoic acid was used to study the impact of α-Syn accumulation on protein phosphorylation and glycosylation. Soluble proteins from 3D5 cells, with or without the induced α-Syn expression were analyzed by two-dimensional gel electrophoresis and staining of gels with dyes that bind to proteins (Sypro ruby), phosphoproteins (Pro-Q diamond) and glycoproteins (Pro-Q emerald). Phosphoproteins were further confirmed by binding to immobilized metal ion affinity column. α-Syn accumulation caused differential phosphorylation and glycosylation of 16 and 12, proteins, respectively, whose identity was revealed by mass spectrometry. These proteins, including HSP90, have diverse biological functions including protein folding, signal transduction, protein degradation and cytoskeletal regulation. Importantly, cells accumulating α-Syn assemblies with different abilities to bind thioflavin S displayed different changes in phosphorylation and glycosylation. Consistent with the cell-based studies, we demonstrated a reduced level of phosphorylated HSP90 α/β in the substantia nigra of subjects with Parkinson's disease as compared to normal controls. Together, the results indicate that α-Syn accumulation causes complex cellular responses, which if persist may compromise cell viability.

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Phosphoproteome profile of human liver Chang's cell based on 2‐DE with fluorescence staining and MALDI‐TOF/TOF‐MS

Cited by 18 publications

References 28 publications

Phosphoproteomic analysis of aged skeletal muscle

Phosphoproteomic analysis of aged skeletal muscle

Quantitative proteomic analysis of lentiviral vectors using 2‐DE

Proteomic profiling of phosphoproteins and glycoproteins responsive to wild-type alpha-synuclein accumulation and aggregation

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