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Regulation of Human CYP2D6
Published in Shufeng Zhou, Cytochrome P450 2D6, 2018
Reversible protein phosphorylation, principally on serine, threonine, or tyrosine residues, is one of the most important and well-studied posttranslational modifications (Beltrao et al. 2012). Phosphorylation plays critical roles in the regulation of many cellular processes including cell cycle, growth, apoptosis, and differentiation. There are limited data on the regulation of CYP2D6 by posttranslational modifications. Using titanium dioxide resin combined with tandem mass spectrometry for phosphopeptide enrichment and sequencing, eight human CYP phosphorylation sites are identified. The data from surgical human liver samples establish that CYP1A2, 2A6, 2B6, 2E1, 2C8, 2D6, 3A4, 3A7, and 8B1 are phosphorylated in vivo (Redlich et al. 2008).
Nonhistone Nuclear Phosphoproteins
Published in Lubomir S. Hnilica, Chromosomal Nonhistone Proteins, 2018
Workers in several laboratories have observed phosphorylated polypeptides of relatively low molecular weight (10,000 to 16,000 daltons) in chromatin. MacGillivray et al.110 found that after labeling mouse liver nuclei with [γ-32P]ATP as much as 10 to 20% of the radioactivity was incorporated into a low molecular weight component of saline-soluble nuclear and chromatin fractions. This polypeptide, which they designated component 10, had a pi near 4.5 and a molecular weight of approximately 10,000. The protein contained both phosphoserine and phosphothreonine and yielded two phosphorylated peptides upon tryptic digestion and phosphopeptide mapping. Although nothing is known about the function of this protein, it was observed in all tissues that were examined by MacGillivray and co-workers,31,32,111 Similar phosphorylated low molecular weight components were reported in rat liver nuclei56,112,113 and in fibroblast chromatin.114
Phosphorylation of Phosphofructokinase — The Possible Role of Covalent Modification in the Regulation of Glycolysis
Published in Rivka Beitner, Regulation of Carbohydrate Metabolism, 1985
The amino terminus of phosphofructokinase, as determined by dansylation and high pressure liquid chromatography after hydrolysis, is phenylalanine.151 The same amino acid was found at the NH2-terminus after mild trypsin treatment of phosphofructokinase. The carboxyl terminus released by carboxypeptidase Y was valine. Short treatment of the enzyme with a low trypsin concentration changed the COOH-terminus to arginine. The labeled phosphopeptides obtained by treatment of phosphofructokinase with subtilisin and trypsin, were subjected to automatic Edman degradation by Kemp and co-workers.59 They found the phosphoserine in a tryptic octapeptide, whose sequence was also present in the dodecapeptide that was released by subtilisin. The sequence of the C-terminal phosphopeptide, as determined by Kemp et al.,59 is
Advances in phosphoproteomics and its application to COPD
Published in Expert Review of Proteomics, 2022
Xiaoyin Zeng, Yanting Lan, Jing Xiao, Longbo Hu, Long Tan, Mengdi Liang, Xufei Wang, Shaohua Lu, Tao Peng, Fei Long
The principle of strong anion exchange (SAX) chromatography is the exact opposite of SCX. The stationary phase of SAX is a positively charged group that binds more strongly to negatively charged phosphopeptides, allowing for better retention of phosphopeptides (Figure 2b). Although acidic phosphopeptides bind well to the exchange resin, phosphopeptide recovery remains a challenge. Dong and coworkers [63] used a SAX capillary column in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to detect phosphopeptides at concentrations down to the amol level. Hardman and colleagues [64] employed an elution gradient at pH 6.0, 6.8 and 8.0, and found that at pH 6.8, the majority of phosphopeptide ion signals from tryptic digests of myoglobin (pHis) peptides can be optimally separated.
Strategies for mass spectrometry-based phosphoproteomics using isobaric tagging
Published in Expert Review of Proteomics, 2021
Xinyue Liu, Rose Fields, Devin K. Schweppe, Joao A. Paulo
The peptides may now either be labeled before phosphopeptide enrichment or enriched prior to labeling (as discussed later). Moreover, once labeled, the peptides can be phosphopeptide-enriched and then fractioned offline or vice versa. For large-scale preparations (>1.5 mg total starting material), samples may be fractionated first and then individual phosphopeptide enrichments may be performed. Phosphopeptides may then be fractionated off-line (for example, with basic pH reversed phase chromatography) and then on-line (with the mass spectrometer) prior to analysis (Figure 1(d)). We note that peptides with the same amino acid sequence (with identical modifications) are chromatographically indistinguishable, regardless of the isobaric tag. These peptides co-elute but as they have the same mass, they will appear as a single composite peak with an identical mass-to-charge (m/z) value in an MS1 scan. The fragmentation of the labeled peptides during the MS2 or MS3 stage generates reporter ion peaks of different masses, whose intensities are used for the relative quantification across samples and downstream bioinformatics analysis [12]. As such, careful sample preparation, including sufficient fractionation is critical for successful phosphopeptide quantification workflows.
Phosphoproteomics: a valuable tool for uncovering molecular signaling in cancer cells
Published in Expert Review of Proteomics, 2021
Jacqueline S. Gerritsen, Forest M. White
High quality/high accuracy phosphopeptide identification and quantification can be critical for defining cancer cell signaling networks from phosphoproteomic data [108]. Since each phosphopeptide is typically a ‘one-hit wonder,’ high stringency in data filtering is recommended to remove as many false positive assignments as possible and to localize the phosphorylation site to the correct residue, if possible. While restrictive filters may compromise the ultimate data set size, high quality data facilitates computational data analysis and ultimate biological insight.