Multiplexing Protein Analysis Core Services

Summary of Method

1. We use an LC-tandem mass spectrometry technique called selected reaction monitoring to measure protein abundance.
2. The assays are designed and validated to target specific proteins based on their amino acid sequence.
3. Targeting gives the assays high sensitivity in complex samples. The assays are precise, accurate, and have a wide dynamic range.
4. Assays for individual proteins are bundled into panels of approximately 40 proteins designed to interrogate selected biochemical pathways (See  Table 1).
5. Our workflow accommodates sufficient sample numbers to design experiments with good statistical power.
6. Our workflow accommodates a wide range of sample types and preparation methods.
7. We are constantly designing new methods and can design methods for new protein targets requested by users.
8. We ask users to submit tissue or cell homogenates, made using tested methods from their lab, with precisely measure protein concentrations. We do the rest of the processing.

Sample Preparation

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.

Data Analysis

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.

Sample Submission

Contact Mike Kinter (mike-kinter@omrf.org) prior to submitting samples.  The goal of the contact will be to develop a detailed plan for the analyses, including:

• goals of the experiment
• experimental design including the number of samples being submitted
• the type and amounts of the samples that will be submitted
• the homogenization procedure being used
• what we will do with samples
• time-frame for completion

Sample Preparation

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.

• Tissue Samples:  The ideal sample is a cleared homogenate prepared in RIPA buffer or something similar. We use 10mL buffer per 150mg of tissue wet weight.  The final protein concentration in the homogenate is approximately 1mg/mL.  These conditions have been used for heart, liver, brain, and eye samples.

• Key factors in the choice of buffers are:

• • Experience your lab has with different buffers is the best place to start.  It is generally best to continue to use the buffer system your lab has the most experience with.  We believe the combination of protein precipitation and short-run gel electrophoresis makes our methods generally tolerant of the wide variety of buffer components that are available.
  • Reducing agents cannot be used.  An accurate protein assay is critical to the overall assay.  We rely on the BioRad DC (detergent compatible) protein assay, which is a variation of the Lowry assay.  It is not compatible with any reducing agents.
  • A good understanding of the extracellular matrix in the tissue being studied is needed.  In some situations, the extracellular matrix has the potential to saturate the samples with large amounts of uninformative protein.  These types of tissues will require additional effort by the submitting lab to develop a homogenization that maximizes the extraction of cellular protein while minimizing the deleterious contribution by the extracellular matrix. 
  • Consistency is critical for precise results.  Because of the potential contribution of factors like the extracellular matrix, the choice of homogenization is important.  Details like the buffer used, presence of detergents, buffer-to-tissue ratio, time, etc. can affect the comparability of the results.

• Cultured Cells: A key difference for cultured cells is the need to wash away the serum proteins in the culture media. Our past experiments wash the monolayer once with Tris buffered saline or phosphate buffered saline. Harvest the cells by scraping into the same buffer, pellet by centrifugation, then resuspend and repellet before lysis.

$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.

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.

Deuterium Oxide:

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.

Sacrifice:

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.

Sample Preparation:

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.

Analysis:

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.

Shipping Samples:

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.

References:

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