Chapter 4. strategies for protein quantitation Ⅱ

Transcription

1 Proteomics Chapter 4. strategies for protein quantitation Ⅱ 1

2 Multiplexed proteomics Multiplexed proteomics is the use of fluorescent stains or probes with different excitation and emission spectra to detect different groups of proteins simultaneously on the same gel. This helps to reduce the number of gels that are required to compare different conditions and at least, in theory, obviate the need for gel matching. 1. Difference gel electrophoresis 2. Parallel analysis with multiple dyes 2

3 Difference gel electrophoresis (DIGE) Different protein samples, eg. healthy vs disease or stimulated vs unstimulated, are labeled on lysine side chains with succinimidyl esters of propyl-cy3 and methyl-cy5 Normal Muscle Proteome Pooled Internal Standard Dystrophic Muscle Proteome Pooled Internal Standard Cy3 Cy5 Cy3 Cy5 2-CyDye DIGE fluors lableing approach Mixed samples Mixed samples * Scanning of 2D IEF/SDS-PAGE separated and co-migrating fluorescently labeled proteins Cy3 image Cy3 image Cy3 image Cy3 image * Image analysis of differential expression pattern of fluorescently labeled proteins 3

4 Difference gel electrophoresis (DIGE) Because the samples run together, all differences in gel preparation, running conditions and local gel structure are eliminated 4

5 Difference gel electrophoresis (DIGE) Drawbacks -Fluorescent labels used are less sensitive than SYPRO dye and silver staining -solubility is lost and the proteins precipitate during electrophoresis -fluorescent conjugate retards the proteins during the SDS- PAGE -protein differ in their labeling efficiency, solubility when conjugated to the label, and the extent to which they might exhibit quenching => Bright and dim spots represent proteins that are present at approximately the same level but show differential labeling efficiency or quenching 5

6 Prallel analysis with multiple dyes The gels stained with SYPRO can also be with additional reagents that identify specific classes of proteins. These proteins can be used as landmarks for gel matching but more importantly the technique can be used to identify subsets of proteins in the proteome that share specific functional attributes -Glycoproteins and phophoproteins -Oligo-histidine tagged proteins -Calcium-binding proteins, -Proteins that have the capability to bind or metabolize drugs 6

7 Parallel analysis with multiple dyes For example, BOCILLIN penicillin analog have been produced carrying BODIPY dyes, which are relatively nonpolar and have a neutral chromophore and therefore do not interfere with the structure or chemical behaviors of the antibiotic. BODIPY Penicillin 7

8 Parallel analysis with multiple dyes This reagent efficiently identify penicillin binding proteins on a 2D-gel with SYPRO Ruby used as a general counterstain. SYPRO BODIPY 8

9 Quantitative proteomics with mass spectrometry Qauntitation can be carried out by comparing peptide ion currents but this is inherently inaccurate and is biased by instruments. The general approach - label alternative samples with equivalent reagents, one of which contains a heavy isotope and one of which contains a light isotope. -The samples are mixed, separated into fractions, and analyzed by mass spectrometry. -The ratio of the two isotopic variants can be determined from the heights of the peaks in the mass spectra and used to identify proteins with differential abundance. Several variants of the approach (Figure 4.3) 9

10 Quantitative proteomics with mass spectrometry Figure

11 ICAT reagents (1) - selective Isotope-coded affinity tag (ICAT) Ex. Biotinylated derivatives, iodoacetamide, a reagent that reacts with the cysteine side chains of denatured proteins Two versions of the reagents are used One normal or light form One heavy or deuterated form in which a hydrogen replced by deuteriun +Variants on this theme include a cleavable ICAT reagent, which allows the biotin to be removed before mass spectrometry. Disadvantage of all the above methods the proteins that do not contain cysteine cannot be quantified 11

12 ICAT reagents (2) Figure

13 Nonselective labeling of peptides after digestion (1) An alternative to the ICAT labeling of proteins As discussed in Chapter 3, when trypsin cleaves a protein and generates a peptide with a new C-terminus, it introduces an oxygen atom derived from a molecule of water into the carboxyl group of the peptide. This can be also used to differentially label peptides derived from alternative protein samples if normal water is used in one buffer and water substituted heavy oxygen ( 18 O) is used on the other. H 2 O + 13

14 Nonselective labeling of peptides after digestion (2) Figure

15 Nonselective labeling of peptides after digestion (3) Alternatives strategies include the chemical modification of the C-terminus, N-terminus or exposed lysine side chains of tryptic peptides using isotope-coded reagent. A hybrid of ICAT and postdigestion labeling strategies involves the use of a solid-phase ICAT reagent to capture cysteine-containing peptides from a complex mixture onto small plastic beads. The reagent photolabile linker arm so that isotope-tagged peptides can be released from the beads by exposure to light. Various coding strategies Table

16 Nonselective labeling of peptides after digestion (3) Table

17 Isotope tagging in vivo (1) In another group of methods, cells treated under different conditions are grown in media containing either normal or heavy isotopes of nitrogen, carbon or hydrogen. A useful approach is the use of labeled amino acids (stableisotope labeling with amino acids in cell culture (SILAC)) The advantage this metabolic labeling approach is that the label is introduced early in the experiment, therefore eliminating variation arising from sample preparation and purification losses. However, it can only be used for the live cells that can be maintained in a controlled environment. (not useful :tissue explants, biopsies, body fluids or cells ) Example: yeast cells were switched from minimal to enriched medium 17

18 Isotope tagging in vivo (2) Figure

19 Mass-coded abundance tags (MCATs) Mass-coded abundance tags, chemical adduct with a specific mass which avoid the need for stable isotopes. Ex. Proteins from the one sample are labeled with O- methylisourea and those from the other sample not labeled. Simple and inexpensive, but less accurate than those involving isotopes. 19