Sample handling for proteome analysis

Staudenmann, Werner; Hatt, Paola Dainese; Hoving, Sjouke; Lehmann, Anton; Kertesz, Michael A.; James, Peter

doi:10.1002/elps.1150190605

Cited by 16 publications

(9 citation statements)

References 14 publications

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“…This feature can potentially increase the sensitivity of mass spectrometric analysis since it minimizes the background chemical noise from upstream sample preparation. The robustness of the ALICE reaction permits the use of high percentage organic solvent throughout the procedure and thus minimizes adsorptive loss, particularly because this procedure avoids a final vacuum concentration of peptide solutions in the presence of water. , The chemical moiety attached to the Cys-containing peptides is small in size and does not fragment under normal low-energy CID conditions. Finally, ALICE has a very high capacity for capturing Cys-containing peptides and the captured peptides are easily eluted off the ALICE resin with high yield using MS-compatible acidic solvents.…”

Section: Discussionmentioning

confidence: 99%

Acid-Labile Isotope-Coded Extractants: A Class of Reagents for Quantitative Mass Spectrometric Analysis of Complex Protein Mixtures

Qiu¹,

Sousa²,

Hewick³

et al. 2002

Anal. Chem.

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Quantitative mass spectrometry using stable isotope-labeled tagging reagents such as isotope-coded affinity tags has emerged as a powerful tool for identification and relative quantitation of proteins in current proteomic studies. Here we describe an integrated approach using both automated two-dimensional liquid chromatography/ mass spectrometry (2D-LC/MS) and a novel class of chemically modified resins, termed acid-labile isotope-coded extractants (ALICE), for quantitative mass spectrometric analysis of protein mixtures. ALICE contains a thiol-reactive group that is used to capture all cysteine (Cys)-containing peptides from peptide mixtures, an acid-labile linker, and a nonbiological polymer. The acid-labile linker is synthesized in both heavy and light isotope-coded forms and therefore enables the direct relative quantitation of peptides/proteins through mass spectrometric analysis. To test the ALICE method for quantitative protein analysis, two model protein mixtures were fully reduced, alkylated, and digested in solution separately and then Cys-containing peptides covalently captured by either light or heavy ALICE. The reacted light and heavy ALICE were mixed and washed extensively under rigorous conditions and the Cys-containing peptides retrieved by mild acid-catalyzed elution. Finally, the eluted peptides were directly subjected to automated 2D-LC/MS for protein identification and LC/MS for accurate relative quantitation. Our initial study showed that quantitation of protein mixtures using ALICE was accurate. In addition, isolation of Cys-containing peptides by the ALICE method was robust and specific and thus yielded very low background in mass spectrometric studies. Overall, the use of ALICE provides improved dynamic range and sensitivity for quantitative mass spectrometric analysis of peptide or protein mixtures.

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Section: Discussionmentioning

confidence: 99%

Acid-Labile Isotope-Coded Extractants: A Class of Reagents for Quantitative Mass Spectrometric Analysis of Complex Protein Mixtures

Qiu¹,

Sousa²,

Hewick³

et al. 2002

Anal. Chem.

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“…The TTK fractions (0.5 mg/5 ml) were partially purified by AF heparin column chromatography [15] and concentrated by 5% SDS–PAGE for 6.5 h at 18 mA in a Y‐shaped gel which has been developed by P. James of the ETH‐Zentrum [21] for mass spectrometry and was modified by us. The main bands between 40 and 28 kDa were separated again by 3%/9% SDS–PAGE for 3 h at 10 mA.…”

Section: Methodsmentioning

confidence: 99%

Tau‐tubulin kinase phosphorylates tau at Ser‐208 and Ser‐210, sites found in paired helical filament‐tau

Tomizawa¹,

Omori²,

Ohtake³

et al. 2001

FEBS Letters

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Hyperphosphorylated tau protein is known to be a major component of the paired helical filaments (PHFs) that accumulate in the brain of Alzheimer's patients. The kinase that phosphorylated Ser-208 and Ser-210 in PHF-tau had remained unknown. We used anti-pS208 and anti-pS210 antibodies and Western blots to confirm that the tau-tubulin kinase (TTK) phosphorylates tau at Ser-208 and at Ser-210. Using partial amino acid sequences of purified bovine brain TTK, a mouse cDNA of TTK was isolated and the sequence was determined. Its 963 bp coding region is composed of 320 amino acids and encodes a 36 kDa protein indistinguishable in size from authentic bovine brain TTK. Our immunoblot analysis demonstrated that TTK is ubiquitously distributed in the rat tissues, and that it is developmentally regulated in the rat brain. ß

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“…As the extensive handling associated with gel-based protein separation can lead to significant sample loss [61], solution-based digestion procedures are often a better choice. The enzyme most commonly used for this purpose is trypsin, largely because of its specificity and tendency to generate peptides which are particularly amenable to sequencing when using positive ionization MS [62].…”

Section: The Proteomics Process For Current Investigations Into Spermmentioning

confidence: 99%

Sperm phosphoproteomics: historical perspectives and current methodologies

Porambo

Salicioni

Visconti³

et al. 2012

Expert Review of Proteomics

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Mammalian sperm are differentiated germ cells that transfer genetic material from the male to the female. Owing to this essential role in the reproductive process, an understanding of the complex mechanisms that underlie sperm function has implications ranging from the development of novel contraceptives to the treatment of male infertility. While the importance of phosphorylation in sperm differentiation, maturation and fertilization has been well established, the ability to directly determine the sites of phosphorylation within sperm proteins and to quantitate the extent of phosphorylation at these sites is a recent development that has relied almost exclusively on advances in the field of proteomics. This review will summarize the work that has been carried out to date on sperm phosphoproteomics and discuss how the resulting qualitative and quantitative information has been used to provide insight into the manner in which protein phosphorylation events modulate sperm function. The authors also present the proteomics process as it is most often utilized for the elucidation of protein expression, with a particular emphasis on the way in which the process has been modified for the analysis of protein phosphorylation in sperm.

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Sample handling for proteome analysis

Cited by 16 publications

References 14 publications

Acid-Labile Isotope-Coded Extractants: A Class of Reagents for Quantitative Mass Spectrometric Analysis of Complex Protein Mixtures

Acid-Labile Isotope-Coded Extractants: A Class of Reagents for Quantitative Mass Spectrometric Analysis of Complex Protein Mixtures

Tau‐tubulin kinase phosphorylates tau at Ser‐208 and Ser‐210, sites found in paired helical filament‐tau

Sperm phosphoproteomics: historical perspectives and current methodologies

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