Targeted Quantitative Proteomics
Selected Reaction Monitoring
Summary of Method
Precise protein concentrations are critical to the overall success of the experiment. The exact amount of protein used will be determined during the pre-experiment discussions with Dr. Kinter or Miller.
In a typical experiment, we take a volume equivalent to 60µg protein and mix with our bovine serum albumin internal standard. The proteins are precipitated with acetone, dissolved in 60µL Laemmli buffer and run 1.5 cm into a SDS-PAGE gel. Each lane is cut as a single sample, the proteins reduced, alkylated, and digested with trypsin. The final peptide mixtures are evaporated to dryness and dissolved in 150µL 1% acetic acid for analysis.
LC-Tandem Mass Spectrometry Analysis
Our laboratory uses a ThermoScientific TSQ Quantiva triple quadrupole mass spectrometry system with an Eksigent nanoflow liquid chromatography system. Five to 10µL of the sample is injected, and the peptides eluted with a linear gradient of acetonitrile in water with 0.1% formic acid. Retention time scheduling is used to maximize the dwell time on each transition. Each analysis takes 80 minutes.
We use the open-source program Skyline to process the data (Michael MacCoss Laboratory at the University of Washington). The initial processing finds and integrates the proper chromatographic peaks for each peptide in the panel. The integration data are exported to Excel to complete the data reporting. Total peptide responses are calculated as the geometric mean of the abundance of each peptide. These responses are then normalized to bovine serum albumin peptides to determine the amount of each measured protein in the sample. The units we use are pmol/100µg of total protein.
Contact Mike Kinter (email@example.com) prior to submitting samples. The goal of the contact will be to develop a detailed plan for the analyses, including:
Our procedures require submission of a tissue or cell homogenate with a precisely assigned protein concentration. The exact amount of protein sent will be determined in the pre-experiment discussions. Ideally, the homogenate is prepared using methods that have been developed and tested in the submitting laboratory.
Samples should be clearly labeled with an informative sample name. We will carry these names through our notes, logs, and reports. Below are examples of methods we have the most experience using.
$60/sample for the first panel of proteins
$30 for each additional panel of proteins.
Approximately $1,000 to develop a panel of 20 proteins.
Synthesis & Turnover
The core will measure the turnover of individual proteins when used in combination with targeted quantitative proteomics.
Impaired protein homeostasis (proteostasis) is a hallmark of aging, and associated with several chronic diseases. Protein turnover, a primary proteostatic mechanism, is a dynamic process that consists of protein synthesis and protein degradation. To assess protein turnover as a proteostatic mechanism, the Core helps in the design and analysis of studies using deuterium oxide (D2O). The use of D2O is advantageous for several reasons: 1) ease of use for many labs, 2) it is relatively cheap, 3) amenable to studies in vitro, in model organisms, and in humans, 4) measurements can span days to weeks, and 5) it allows for secondary measurements including DNA and RNA synthesis, which have proteostatic implications.
Monitoring D2O incorporation into amino acids, peptides, or nucleic acids over time allows for the determination of synthetic rates of proteins, RNA and DNA. In animals and humans, D2O is provided in the drinking water, while for in vitro studies D2O is added to the culture media. Deuterium rapidly equilibrates with the body water pool, and is incorporated into non-labile sites on amino acids, ribose and deoxyribose (precursors for protein and DNA). After the labeling period, tissues or cells are harvested, homogenized, and prepared for proteomic, GC-MS, or GC-QQQ analysis. Calculations of synthesis rates are based on the precursor-product relationship and requires the enrichment of the product of interest and the precursor that gives rise to the product.
Most studies use either 99% enriched D2O (Sigma Aldrich 756822) that may be diluted to 70%.
It is imperative to consult with Dr. Miller prior to performing labeling experiments since the study design differs based on the information desired.
D2O labeling methods can be used in vivo (animals, humans) and in vitro. The steps outlined below describe a basic approach for a labeling experiment in mice. These methods are also adaptable for studies in vitro, in model organisms, and in humans:
Bolus Intraperitoneal Injection of D2O:
The I.P. injection is given at the start of the experiment to bring body water enrichment up to target enrichment rapidly. For the I.P. injection, sterile filter 99% D2O (with 0.9% NaCl (w/v) added) using a .22 micron filter, and administer a bolus injection based on the body weight of the animal. To achieve the target enrichment of 5%, mice are given an injection based on 60% of the body weight as water.
Ad Libitum Drinking Water:
Drinking water that is enriched with D2O is used to maintain steady state of body water enrichment for the duration of the study. The D2O enriched water is made by diluting 99% D2O (stock) with normal tap water.
At sacrifice, the tissues of interested are harvested and snap frozen. In addition, we require plasma and bone marrow (for nucleic acids) for the determination of precursor enrichment.
Samples for proteomic analysis will follow basic prep as described in for LC-MS (link to Kinters methods). We recommend the Qiagen QIAamp DNA Mini Kit for DNA extraction or a Trizol extraction procedure for RNA. Follow directions for high quality nucleic acid extraction.
Proteomic analysis will use methods established in the Multiplexing Analysis Core (link to Kinter’s methods). DNA and RNA will be analyzed using GC-MS or GC-QQQ.
When sending sample boxes please label the tubes clearly, and organize them in a logical format within the box (chronologically, by group, by sex, labeling timepoint, etc). An excel sheet listing all of the samples included in the box, and descriptions of all acronyms used is also helpful.
Miller, B. F., Baehr, L. M., Musci, R. V., Reid, J. J., Peelor, F. F., III, Hamilton, K. L., & Bodine, S. C. (2019a). Muscle‐specific changes in protein synthesis with aging and reloading after disuse atrophy. Journal of Cachexia, Sarcopenia and Muscle, 34, 24–15. http://doi.org/10.1002/jcsm.12470
Miller, B. F., Hamilton, K. L., Majeed, Z. R., Abshire, S. M., Confides, A. L., Hayek, A. M., et al. (2018). Enhanced skeletal muscle regrowth and remodelling in massaged and contralateral non-massaged hindlimb. The Journal of Physiology, 596(1), 83–103. http://doi.org/10.1113/JP275089
Miller, B. F., Pharaoh, G. A., Hamilton, K. L., Peelor, F. F., Kirkland, J. L., Freeman, W. M., et al. (2019b). Short-term calorie restriction and 17α-estradiol administration elicit divergent effects on proteostatic processes and protein content in metabolically active tissues. The Journals of Gerontology Series a: Biological Sciences and Medical Sciences. http://doi.org/10.1093/gerona/glz113
Robinson, M. M., Turner, S. M., Hellerstein, M. K., Hamilton, K. L., & Miller, B. F. (2011). Long-term synthesis rates of skeletal muscle DNA and protein are higher during aerobic training in older humans than in sedentary young subjects but are not altered by protein supplementation. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 25(9), 3240–3249. http://doi.org/10.1096/fj.11-186437
Scalzo, R. L., Peltonen, G. L., Binns, S. E., Shankaran, M., Giordano, G. R., Hartley, D. A., et al. (2014). Greater muscle protein synthesis and mitochondrial biogenesis in males compared with females during sprint interval training. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 28(6), 2705–2714. http://doi.org/10.1096/fj.13-246595
Protein synthesis: $50/ sample
D2O labeling: $35/sample
Isotopic enrichment of DNA and RNA: $65/sample
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