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248
Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution * □S
"... Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimize ..."
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Cited by 58 (3 self)
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Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1–10 ng/ml range with percent coefficients of variation from 3 to 15 % without immunoaffinity enrichment of either proteins
Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF
- J. Proteome Res
, 2006
"... A comparative study on the three quantitative methods frequently used in proteomics, 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. DIGE and cICAT are familiar techniques use ..."
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Cited by 54 (1 self)
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A comparative study on the three quantitative methods frequently used in proteomics, 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. DIGE and cICAT are familiar techniques used in gel- and LC-based quantitative proteomics, respectively. iTRAQ is a new LC-based technique which is gradually gaining in popularity. A systematic comparison among these quantitative methods has not been reported. In this study, we conducted well-designed comparisons using a six-protein mixture, a reconstituted protein mixture (BSA spiked into human plasma devoid of six abundant proteins), and complex HCT-116 cell lysates as the samples. All three techniques yielded quantitative results with reasonable accuracy when the six-protein or the reconstituted protein mixture was used. In DIGE, accurate quantification was sometimes compromised due to comigration or partial comigration of proteins. The iTRAQ method is more susceptible to errors in precursor ion isolation, which could be manifested with increasing sample complexity. The quantification sensitivity of each method was estimated by the number of peptides detected for each protein. In this regard, the global-tagging iTRAQ technique was more sensitive than the cysteine-specific cICAT method, which in turn was as sensitive as, if not more sensitive than, the DIGE technique. Protein profiling on HCT-116 and HCT-116 p53-/cell lysates displayed limited overlapping among proteins identified by the three methods, suggesting the complementary nature of these methods. Keywords: protein quantification • DIGE • cICAT • iTRAQ • 2D gel • LC-MALDI TOF/TOF
Proteomics discovery of metalloproteinase substrates in the cellular context by iTRAQ labeling reveals a diverse MMP-2 substrate degradome
- Mol. Cell. Proteomics
, 2007
"... Elucidation of protease substrate degradomes is essen-tial for understanding the function of proteolytic pathways in the protease web and how proteases regulate cell func-tion. We identified matrix metalloproteinase-2 (MMP-2) cleaved proteins, solubilized pericellular matrix, and shed cellular ectod ..."
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Cited by 30 (5 self)
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Elucidation of protease substrate degradomes is essen-tial for understanding the function of proteolytic pathways in the protease web and how proteases regulate cell func-tion. We identified matrix metalloproteinase-2 (MMP-2) cleaved proteins, solubilized pericellular matrix, and shed cellular ectodomains in the cellular context using a new multiplex proteomics approach. Tryptic peptides of intact and cleaved proteins, collected from conditioned culture medium of Mmp2/ fibroblasts expressing low levels of transfected active human MMP-2 at different time points, were amine-labeled with iTRAQTM mass tags. Peptide identification and relative quantitation between active and inactive protease transfectants were achieved following tag fragmentation during tandem MS. Known substrates of MMP-2 were identified thereby validating this tech-
Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins
- Curr. Biol. 2007
"... Background: The nucleolus is a subnuclear organelle in which rRNAs are transcribed, processed, and assembled with ribosomal proteins into ribosome subunits. Mass spectrometry combined with pulsed incorporation of stable isotopes of arginine and lysine was used to perform a quantitative and unbiased ..."
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Cited by 24 (3 self)
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Background: The nucleolus is a subnuclear organelle in which rRNAs are transcribed, processed, and assembled with ribosomal proteins into ribosome subunits. Mass spectrometry combined with pulsed incorporation of stable isotopes of arginine and lysine was used to perform a quantitative and unbiased global analysis of the rates at which newly synthesized, endogenous proteins appear within mammalian nucleoli. Results: Newly synthesized ribosomal proteins accumulated in nucleoli more quickly than other nucleolar components. Studies involving time-lapse fluorescence microscopy of stable HeLa cell lines expressing fluorescent-protein-tagged nucleolar factors also showed that ribosomal proteins accumulate more quickly than other components. Photobleaching and mass-spectrometry experiments suggest that only a subset of newly synthesized ribosomal proteins are assembled into ribosomes and exported to the cytoplasm. Inhibition of the proteasome caused an accumulation of ribosomal proteins in the nucleus but not in the cytoplasm. Inhibition of rRNA transcription prior to proteasomal inhibition further increased the accumulation of ribosomal proteins in the nucleoplasm. Conclusions: Ribosomal proteins are expressed at high levels beyond that required for the typical rate of ribosome-subunit production and accumulate in the nucleolus more quickly than all other nucleolar components. This is balanced by continual degradation of unassembled ribosomal proteins in the nucleoplasm, thereby providing a mechanism for mammalian cells to ensure
Identifying dynamic interactors of protein complexes by quantitative mass spectrometry
- Mol Cell Proteomics
"... Dynamically interacting proteins associate and dissociate with their binding partners at high on/off rates. Although their identification is of great significance to proteomics research, lack of an efficient strategy to distinguish stable and dynamic interactors has hampered the efforts toward this ..."
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Cited by 21 (0 self)
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Dynamically interacting proteins associate and dissociate with their binding partners at high on/off rates. Although their identification is of great significance to proteomics research, lack of an efficient strategy to distinguish stable and dynamic interactors has hampered the efforts toward this goal. In this work, we developed a new method, MAP (mixing after purification)-SILAC (stable isotope labeling of amino acids in cell culture), to quantitatively investigate the interactions of protein complexes by mass spectrom-etry. In combination with the original SILAC approach, stable and dynamic components were effectively distin-guished by the differences in their relative abundance ratio changes. We applied the newly developed strategies to decipher the dynamics of the human 26 S proteasome-interacting proteins. A total of 67 putative human protea-
Eight-channel itraq enables comparison of the activity of 6 leukaemogenic tyrosine kinases
- Mol. Cell Proteomics
, 2007
"... tyrosine kinases. ..."
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Robust and Sensitive iTRAQ Quantification on an LTQ Orbitrap Mass Spectrometer*□S
"... Isobaric stable isotope tagging reagents such as tandem mass tags or isobaric tags for relative and absolute quan-tification enable multiplexed quantification of peptides via reporter ion signals in the low mass range of tandem mass spectra. Until recently, the poor recovery of low mass fragments ob ..."
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Cited by 18 (1 self)
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Isobaric stable isotope tagging reagents such as tandem mass tags or isobaric tags for relative and absolute quan-tification enable multiplexed quantification of peptides via reporter ion signals in the low mass range of tandem mass spectra. Until recently, the poor recovery of low mass fragments observed in tandem mass spectra ac-quired on ion trap mass spectrometers precluded the use of these reagents on this widely available instrument plat-form. The Pulsed Q Dissociation (PQD) technique allows negotiating this limitation but suffers from poor fragmen-tation efficiency, which has raised doubts in the commu-nity as to its practical utility. Here we show that by care-fully optimizing instrument parameters such as collision energy, activation Q, delay time, ion isolation width, num-ber of microscans, and number of trapped ions, low m/z
Quantitative proteomics by metabolic labeling of model organisms
- Mol Cell Proteomics
"... In the biological sciences, model organisms have been used for many decades and have enabled the gathering of a large proportion of our present day knowledge of basic biological processes and their derailments in disease. Al-though in many of these studies using model organisms, the focus has primar ..."
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Cited by 16 (0 self)
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In the biological sciences, model organisms have been used for many decades and have enabled the gathering of a large proportion of our present day knowledge of basic biological processes and their derailments in disease. Al-though in many of these studies using model organisms, the focus has primarily been on genetics and genomics approaches, it is important that methods become avail-able to extend this to the relevant protein level. Mass spectrometry-based proteomics is increasingly becoming the standard to comprehensively analyze proteomes. An important transition has been made recently by moving from charting static proteomes to monitoring their dy-namics by simultaneously quantifying multiple proteins obtained from differently treated samples. Especially the labeling with stable isotopes has proved an effective
Quantitative profile of five murine core proteomes using label-free functional proteomics.Mol
- Cell. Proteomics
, 2007
"... Analysis of primary animal and human tissues is key in biological and biomedical research. Comparative pro-teomics analysis of primary biological material would benefit from uncomplicated experimental work flows ca-pable of evaluating an unlimited number of samples. In this report we describe the ap ..."
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Cited by 15 (1 self)
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Analysis of primary animal and human tissues is key in biological and biomedical research. Comparative pro-teomics analysis of primary biological material would benefit from uncomplicated experimental work flows ca-pable of evaluating an unlimited number of samples. In this report we describe the application of label-free pro-teomics to the quantitative analysis of five mouse core proteomes. We developed a computer program and nor-malization procedures that allow exploitation of the quan-titative data inherent in LC-MS/MS experiments for rela-tive and absolute quantification of proteins in complex mixtures. Important features of this approach include (i) its ability to compare an unlimited number of samples, (ii) its applicability to primary tissues and cultured cells, (iii) its straightforward work flow without chemical reaction
Accurate quantification of more than 4,000 mouse tissue proteins reveals minimal proteome changes during aging. Mol. Cell Proteomics
, 2011
"... The biological process of aging is believed to be the result of an accumulation of cellular damage to biomol-ecules. Although there are numerous studies address-ing mutation frequencies, morphological or transcrip-tional changes in aging mammalian tissues, few have measured global changes at the pro ..."
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Cited by 12 (0 self)
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The biological process of aging is believed to be the result of an accumulation of cellular damage to biomol-ecules. Although there are numerous studies address-ing mutation frequencies, morphological or transcrip-tional changes in aging mammalian tissues, few have measured global changes at the protein level. Here, we present an in depth proteomic analysis of three brain regions as well as heart and kidney in mice aged 5 or 26 months, using stable isotope labeling of whole animals (SILAC mouse) and high resolution mass spectrometry. In the frontal cortex and hippocampal regions of the brain, more than 4200 proteins were quantitatively com-pared between age groups. Proteome differences be-tween individual mice were observable within and be-tween age groups. However, mean protein abundance
