Elisabeth Huff Lonergan, PhD Steven M. Lonergan, PhD Mark J. Anderson, PhD

Transcription

1 Elisabeth Huff Lonergan, PhD Steven M. Lonergan, PhD Mark J. Anderson, PhD

2 The complexity of tenderness. Differences in tenderness due to postmortem aging is difficult to predict and manage Structure is complex Diversity in protein isoforms between muscles adds another layer of complexity Application of tools to uncover the mechanism of tenderness 2-D DIGE Protein ID methods Mass spectroscopy Immuno techniques Protein Staining Diagonal PAGE

3 2-D Gel 250 pi 3 10 Molecular Weight (kda) 20 Separation of proteins in 2-dimensions

4 Application of 2D DIGE in Meat Science Sarcoplasmic Proteins Myofibrillar Proteins Green = 1 day postmortem (Cy3 dye) Red = 14 days postmortem (Cy5 dye) Yellow = Overlaid spots Mark Anderson, 2009

5 CyDyes Fluorescent dyes that are detected at different wavelengths of light. similar molecular weights (~450 Da) same charge (+1) attach to the lysine residues on proteins Most commonly used CyDyes Cy2 Cy3 Cy5

6 2D Difference In-Gel Electrophoresis (DIGE) Samples Sample 1 Label with Cy3 Labeled Proteins Mixture Migrates in 2D electrophoresis IPG Strip Label with Cy5 Sample 2 Mix Label with Cy2 Pooled Internal Standard SDS PAGE Adapted from Posch, A. Ed Sample preparation for 2D PAGE. Methods in Molecular Biology.

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8 Analysis DeCyder 2D software v6.5 GE Healthcare, Piscataway, NJ Pick spots of interest Electrospray Ionization Mass Spectroscopy

9 2-D DIGE Comparison of Steaks Differing in Star Probe Value To identify proteins in bovine longissimus dorsi muscle that are related to tenderness. Sarcoplasmic fraction was extracted from steaks aged one day postmortem. 50mM Tris, 1mM EDTA, ph D DIGE four comparisons were made between the high star probe (HSP) samples and the low star probe (LSP) samples

10 2-D DIGE Comparisons Comparison 1 Comparison 2 Comparison 3 Cy 2 Cy 3 Cy 5 Internal Standard LSP 1 HSP 1 Internal Standard HSP 2 LSP 1 Internal Standard LSP 2 HSP 2 Comparison 4 Internal Standard HSP 1 LSP 2

11 MW (kda) 4 pi 7 Myosin light chain 1 (MLC1) Phosphoglucomutase- 1 (PGM1) 37 25

12 Proteins that may be potential indicators of tenderness?? Fraction Greater abundance in Actin Sarcoplasmic LSP Tropomyosin alpha-1 chain Sarcoplasmic LSP Myomesin-2 Sarcoplasmic LSP Phosphoglucomutase 1 (4 spots) Sarcoplasmic LSP Phosphoglucomutase 1 (1 spot) Sarcoplasmic HSP Myosin light chain 1 Sarcoplasmic LSP

13 Phosphoglucomutase 1 2D DIGE results identified phosphoglucomutase 1 (PGM1) as differing between HSP and LSP samples. Most alkaline form was most abundant in HSP samples Enzyme involved in glycogen metabolism PGM1 undergoes posttranslational modifications Phosphorylation Acetylation Methylation PGM1 is activated by phosphorylation of a threonine residue. Posttranslational modifications regulate the function of many intercellular proteins in vivo. These modifications can affect: cellular locations signaling pathways protein activity Gururaj et al. (2004). Oncogene, 23(49), Bouley et al. (2004). Rencontre Recherches Ruminants, 11,

14 A closer look at Phosphoglucomutase-1 Sarcoplasmic fraction was extracted from HSP and LSP samples. 900 μg of sarcoplasmic protein extracted from either the samples from HSP steaks or LSP steaks was loaded onto each gel.

15 Phosphoglucomutase - 1 Gels were stained for phosphorylated protein using ProQ Diamond and imaged. Gels were then stained for total protein using Sypro Ruby and imaged. Gels were analyzed to determine the intensity (spot volume) of individual phosphoglucomutase 1 isoforms using the DeCyder 2D Software

16 Identification of Phosphoglucomutase Total Protein Stain Western Blot for PGM1

17 Phosphorylated protein in samples from HSP and LSP steaks High Star Probe Low Star Probe Pro Q Diamond Stain

18 Ratio of phosphorlyated protein Comparison between the HSP and LSP in abundance of phosphoglucomutase 1 isoforms in gels stained for phosphorylated protein PGM1 isoform number * HSP *- Indicates significant difference (P < 0.05) between the HSP and LSP samples HSP High star probe samples LSP Low star probe samples

19 Implications Glycogen Phosphoglucomutase-1 Glucose Glucose-1-phosphate Glucose-6-phosphate Phospoglucomutase-1 activated through phosphorylation Could it affect ph decline? Early rate of ph decline has been shown to affect tenderness of beef. Glycolysis ATP Production Lactic Acid

20 MW (kda) 4 pi 7 Myosin light chain 1 (MLC1) Phosphoglucomutase- 1 (PGM1) 37 25

21 Myosin Light Chain 1 A B b a Myosin S1 Actin b a Hayashibara and Miyanishi Biochemistry 33:

22 A) Troponin-T western blots run on 15% acrylamide separating gel loaded with 60 μg of protein from the pellet collected after the incubation procedure. Densitometry measurements were made on the bands corresponding to the 30 kda degradation product of troponin-t. B) The intensity of 30 kda troponin-t band from western blots preformed on the pellet for each incubation treatment. The ratio shown is the intensity of the 30 kda troponin-t band to the intensity of the same band in a reference sample (longissimus dorsi, aged 7 day postmortem), which appeared on all blots, was used to normalize comparisons across all blots. a-b indicates significant differences at P < n=3

23 A) Myosin light chain 1 western blots run on 15% acrylamide separating gel loaded with all of the protein collected from the supernatant after the incubation procedure. Densitometry measurements were made on the bands corresponding to the 21 kda band of MLC1. n=3 B) The intensity of ~20 kda myosin light chain 1 band from western blots preformed on the supernatant for each incubation treatment. The ratio shown is the intensity of the ~20 kda myosin light chain 1 band to the intensity of the same band in a reference sample (longissimus dorsi, aged 7 day postmortem), which appeared on all blots, was used to normalize comparisons across all blots. a- b indicates significant differences at P < 0.05.

24 Conclusions μ-calpain catalyzes the rapid release of MLC1 from the myofibril into the highly soluble fraction. The rapid release of MLC1 from the myofibril may provide an early indication of the amount of postmortem tenderization that will occur.

25 Other Proteomics Techniques There IS more to life than 2-D gels

26 Identifying protein interactions Calpain interacts with numerous constituents in the cell Calpastatin Cell membranes What else?

27 Immunoprecipitate with µ and m Calpain Confirm with immunoprecipitation with putative binding partner (Ryr1) Identify putative binding partners with MS Brule et al., Biochimie. 92:

28 Ca 2+ Release From Sarcoplasmic Reticulum Ryanodine receptor Ca 2+ release channel in the sarcoplasmic reticulum Activated by submicromolar Ca 2+ and millimolar ATP. (Hurwitz, L. et al eds. Calcium Channels:Their Properties, Functions, Regulation, and Clinical Relevance ) Substrate for µ-calpain (Brandt et al J. Mem. Biol. 127:35-47) McPherson et al J Biol Chem. 268(19):

29 Ryanodine Receptor Degradation (24 h) Associated With Greater µ-calpain Autolysis Pig # C Intact Band A µ-calpain 24 h WBS 21 d (Kg) kda 78 kda 76 kda

30 µ-calpain Autolysis and Proteolysis Increased autolysis related to increased proteolysis. 24 h postmortem Western blot µ-calpain catalytic subunit A B 24 h postmortem Western blot Troponin-T A B 80 kda 78 kda 76 kda } Troponin-T 30 kda ph at 2 hours PM ph at 24 hours PM Rowe et al J. Anim. Sci 79(Suppl. 1):20

31 Identifying protein interactions Protein Oxidation and Protein Cross-linking HiOx-MAP (80% O % CO 2 ) allows: more oxygen to penetrate into meat, consequently forming a higher percentage of oxymyoglobin and a brighter cherry red meat color CO 2 in the package preventing microbial growth HiOx-MAP system creates oxidative conditions HiOx-MAP (80% O % CO 2 ) are likely to increase the incidence of oxidative changes in the meat, and thus it may negatively affect meat quality characteristics.

32 What Is happening to the proteins during oxidizing conditions? Trim & cut a steak (2.54 cm) LD AD HiOx-MAP VAC Displayed for 9d at 1 C - Sensory, biochemical analysis (SDS-PAGE, Western blot Myosin heavy chain, & Diagonal PAGE

33 Oxidizing condition (HiOx-MAP) decreased meat tenderness Sensory Tenderness a b c c c c MAP VAC A. SDS-PAGE 2 0 LL SM AD Sensory Chewiness SEM = c c c c a b MAP VAC B. Western blot of X-linked MHC 0 LL SM AD SEM = 0.33 Kim, Y. H., E. Huff-Lonergan, J. G. Sebranek, and S. M. Lonergan Meat Science 85:

34 Reducing condition Non-reducing condition Reducing condition Diagonal-PAGE to determine intermolecular cross-linked protein 2 nd Dimension SDS-PAGE LV d9 LM d9 LV d9 LM d9 Non-reducing condition First dimension SDS-PAGE Intermolecular disulfide bonds

35 Cross-linking of MHC with Titin? Oxidizing condition resulted in protein polymerization A Titin B Titin MHC MHC MHC VAC MAP

36 In Summary. Proteomics can aid in identifying elusive factors like: Posttranslational modifications Protein-protein interactions Identifying small changes in proteins Must continue to use cutting edge technologies to our advantage BUT true advancement requires: Careful experimental design Collaboration Sharing of data across research groups Cooperation and innovation needed if we are to power forward

37 Acknowledgements Dr. Ed Steadham Trisha Grevengoed Aaron Asmus Dr. Yuan (Brad) Kim Funding The Beef Checkoff Pork Checkoff USDA NRICGP

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