Controlling Deuterium Isotope Effects in Comparative Proteomics

Zhang, Roujian; Sioma, Cathy; Thompson, Robert A.; Li, Xiong; Regnier, Fred E.

doi:10.1021/ac025614w

Cited by 235 publications

(238 citation statements)

References 25 publications

(47 reference statements)

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“…Amplification of isotope effects is a potential disadvantage for high-density labeling. This phenomenon has been observed for some isotopically labeled alkylating agents (77)(78)(79). We observed no isotope effect on MT peptides with 15 N-versus 14 N-carbamidomethylation.…”

Section: Discussionsupporting

confidence: 81%

Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins

Mehus

Muhonen

Garrett

et al. 2014

Molecular & Cellular Proteomics

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Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with 14 N-or 15 N-iodoacetamide. Absolute quantitation was achieved using 15 Niodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue-and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptornegative cell lines than in estrogen-receptor-positive cells. These findings demonstrate that individual human MT isoforms can be accurately quantified in cells and tissues at the protein level, complementing and expanding mRNA measurement as a means for evaluating MTs as potential biomarkers for cancers or heavy metal toxicity. Molecular & Cellular

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Section: Discussionsupporting

confidence: 81%

Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins

Mehus

Muhonen

Garrett

et al. 2014

Molecular & Cellular Proteomics

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“…Sodium phosphate, dibasic anhydrous (Na 2 HPO 4 ), and sodium phosphate monobasic anhydrous (NaH 2 PO 4 ) were from Amresco. The 4-trimethylammoniumbutyryl (TMAB) stable isotopic labeling reagents containing either three, six, and nine atoms of deuterium (D3, D6, and D9-TMAB) or no deuterium (D0-TMAB) were synthesized as described (29)(30)(31) Mice. TAP1/β2m double double-knockout mice were originally provided by Luc Van Kaer from Vanderbilt University School of Medicine Nashville, USA.…”

Section: Methodsmentioning

confidence: 99%

Similar Intracellular Peptide Profile of TAP1/β2 Microglobulin Double-Knockout Mice and C57BL/6 Wild-Type Mice as Revealed by Peptidomic Analysis

Castro

Berti²,

Russo³

et al. 2010

AAPS J

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Abstract. Cells produce and use peptides in distinctive ways. In the present report, using isotope labeling plus semi-quantitative mass spectrometry, we evaluated the intracellular peptide profile of TAP1/β2m −/− (transporter associated with antigen-processing 1/ß2 microglobulin) double-knockout mice and compared it with that of C57BL/6 wild-type animals. Overall, 92 distinctive peptides were identified, and most were shown to have a similar concentration in both mouse strains. However, some peptides showed a modest increase or decrease (~2-fold), whereas a glycine-rich peptide derived from the C-terminal of neurogranin (KGPGPGGPGGAGGARGGAGGGPSGD) showed a substantial increase (6-fold) in TAP1/β2m −/− mice. Thus, TAP1 and β2microglobulin have a small influence on the peptide profile of neuronal tissue, suggesting that the presence of peptides derived from intracellular proteins in neuronal tissue is not associated with antigens of the class I major histocompatibility complex. Therefore, it is possible that these intracellular peptides play a physiological role.

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“…Another methodological consideration is the possible deuterium isotope effect reflected in the resolution of 2 H-labeled and unlabeled peptides caused by chromatographic fractionation (34). Such fractionation depends on mass difference between unlabeled and 2 H-labeled molecules, as was previously observed for 2 H-coded peptides when 2 H 8 -ICAT reagent was used in comparative proteomics (35).…”

Section: Plasma Proteome Dynamicsmentioning

confidence: 96%

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

Willard

Rachdaoui

et al. 2012

Molecular & Cellular Proteomics

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Understanding the pathologies related to the regulation of protein metabolism requires methods for studying the kinetics of individual proteins. We developed a 2 H 2 O metabolic labeling technique and software for protein kinetic studies in free living organisms. This approach for proteome dynamic studies requires the measurement of total body water enrichments by GC-MS, isotopic distribution of the tryptic peptide by LC-MS/MS, and estimation of the asymptotical number of deuterium incorporated into a peptide by software. We applied this technique to measure the synthesis rates of several plasma lipoproteins and acute phase response proteins in rats. Samples were collected at different time points, and proteins were separated by a gradient gel electrophoresis.2 H labeling of tryptic peptides was analyzed by ion trap tandem mass spectrometry (LTQ MS/MS) for measurement of the fractional synthesis rates of plasma proteins. The high sensitivity of LTQ MS in zoom scan mode in combination with 2 H label amplification in proteolytic peptides allows detection of the changes in plasma protein synthesis related to animal nutritional status. Our results demonstrate that fasting has divergent effects on the rate of synthesis of plasma proteins, increasing synthesis of ApoB 100 but decreasing formation of albumin and fibrinogen. We conclude that this technique can effectively measure the synthesis of plasma proteins and can be used to study the regulation of protein homeostasis under physiological and pathological conditions.

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Controlling Deuterium Isotope Effects in Comparative Proteomics

Cited by 235 publications

References 25 publications

Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins

Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins

Similar Intracellular Peptide Profile of TAP1/β2 Microglobulin Double-Knockout Mice and C57BL/6 Wild-Type Mice as Revealed by Peptidomic Analysis

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

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