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Mass spec using

Although GGMS is the most widely used ana lytical method that combines a chromatographic sep aration with the identification power of mass spectrometry it is not the only one Chemists have coupled mass spectrometers to most of the mstru ments that are used to separate mixtures Perhaps the ultimate is mass spectrometry/mass spectrome try (MS/MS) m which one mass spectrometer gener ates and separates the molecular ions of the components of a mixture and a second mass spec trometer examines their fragmentation patterns ... [Pg.573]

An alternative approach to peptide sequencing uses a dry method in which the whole sequence is obtained from a mass spectrum, thereby obviating the need for multiple reactions. Mass spec-trometrically, a chain of amino acids breaks down predominantly through cleavage of the amide bonds, similar to the result of chemical hydrolysis. From the mass spectrum, identification of the molecular ion, which gives the total molecular mass, followed by examination of the spectrum for characteristic fragment ions representing successive amino acid residues allows the sequence to be read off in the most favorable cases. [Pg.333]

Fig. 3.3. Experimental arrangement used by Krauss and Gruen for SSIMS [3.8] a qua-drupole mass spectrometer was used for mass analysis and a retarding-field analyzer for prior energy selection (a) ion gun (b)-(d) lenses 1-3 (e) quadrupole mass spec-... Fig. 3.3. Experimental arrangement used by Krauss and Gruen for SSIMS [3.8] a qua-drupole mass spectrometer was used for mass analysis and a retarding-field analyzer for prior energy selection (a) ion gun (b)-(d) lenses 1-3 (e) quadrupole mass spec-...
Modern reviews of BDEs in closed-shell molecules measured by using mass spec-trometric methods have been provided by Ellison and co-workers. ° Additional evaluations have been provided by Ervin and DeTuri. A list of important fundamental hydrocarbon BDEs is given in Table 5.2. [Pg.220]

ICAT reagents can be used to compare two different samples by mass spec analysis. For instance, one cell population can be treated with a drug candidate, while another one remains untreated and acts as a control. Alternatively, one cell population can represent a disease state and the control population is the normal cell line. After cell lysis, the proteins in each... [Pg.652]

Figure 16.4 A more advanced ICAT design uses an acid-cleavable spacer arm to facilitate elution of labeled peptides from a (strept)avidin affinity column. The use of 14C isotopes instead of deuterium labels permits precise reverse phase separations prior to mass spec that show no elution peak time differences between isotope-labeled and normal atom-labeled peptides. Figure 16.4 A more advanced ICAT design uses an acid-cleavable spacer arm to facilitate elution of labeled peptides from a (strept)avidin affinity column. The use of 14C isotopes instead of deuterium labels permits precise reverse phase separations prior to mass spec that show no elution peak time differences between isotope-labeled and normal atom-labeled peptides.
Figure 16.5 A catch-and-release ICAT design incorporates a gem-methyl group and an isopropyl group on either side of a disulfide bond within its spacer arm. The hindered disulfide permits the use of standard reducing gel electrophoresis conditions using DTT without reduction. After purification on a (strept)avidin affinity column, however, the disulfide group can be cleaved with TCEP, which provides recovery of the labeled peptides prior to mass spec separation. Figure 16.5 A catch-and-release ICAT design incorporates a gem-methyl group and an isopropyl group on either side of a disulfide bond within its spacer arm. The hindered disulfide permits the use of standard reducing gel electrophoresis conditions using DTT without reduction. After purification on a (strept)avidin affinity column, however, the disulfide group can be cleaved with TCEP, which provides recovery of the labeled peptides prior to mass spec separation.
The O-ECAT reagent is a superior alternative to the use of 2,4-dinitrophenylhydrazine (DNPH Chapter 1, Section 1.1) in the study of protein oxidation. DNPH modification produces detectable complexes, but it does not provide information as to what amino acids are involved. O-ECAT modifies carbonyl end products of protein oxidation and in addition, it can provide exact information as to the amino acids that were oxidized. Mass spec analysis of modified proteins performed after proteolysis gives the exact amino acid sequences including the sites of O-ECAT reagent modification. The same antibody that is specific for the metal chelate portion of the standard ECAT reagent also can be used to capture and detect the O-ECAT... [Pg.658]

Figure 18.1 A trifunctional reagent for studying protein interactions by mass spec. The bis-NHS ester arms crosslink interacting proteins, while the discrete PEG-containing biotin arm can be used to isolate or detect the conjugates using (strept)avidin reagents. Figure 18.1 A trifunctional reagent for studying protein interactions by mass spec. The bis-NHS ester arms crosslink interacting proteins, while the discrete PEG-containing biotin arm can be used to isolate or detect the conjugates using (strept)avidin reagents.
Guerrero et al. (2006) used this technique along with the quantitative mass spec strategy called SILAC (stable isotope labeling of amino acids in cell culture Ong et al., 2002) to identify the yeast proteins that interact with the 26 S proteasome. [Pg.1011]

Figure 28.5 This PIR compound contains NHS esters at both ends to capture interacting proteins through amide bond formation. It also contains MS cleavable bonds that release a central reporter group, which can be used to identify crosslinked peptides by mass spec. Figure 28.5 This PIR compound contains NHS esters at both ends to capture interacting proteins through amide bond formation. It also contains MS cleavable bonds that release a central reporter group, which can be used to identify crosslinked peptides by mass spec.
The use of PIR compounds to study protein interactions is a significant advance over the use of standard homobifunctional crosslinkers. The unique design of the PIR reagent facilitates deconvolution of putative protein interaction complexes through a simplified mass spec analysis. The software can ignore all irrelevant peak data and just focus analysis on the two labeled peptide peaks, which accompany the reporter signal of appropriate mass. This greatly simplifies the bioinformatics of data analysis and provides definitive conformation of protein-protein crosslinks. [Pg.1015]


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Mass spec

SPECS

Using mass

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