| Metadata |
| datasetIdentifier | PASS00895 |
| datasetType | SRM |
| submitter | Stephen Holman <stephen.holman@liverpool.ac.uk> |
| submitter_organization | University of Liverpool |
| lab_head_full_name | Professor Robert J. Beynon |
| lab_head_email | r.beynon@liv.ac.uk |
| lab_head_organization | University of Liverpool |
| lab_head_country | UK |
| datasetTag | UoL_turnover_SRM |
| datasetTitle | Protein turnover measurement using selected reaction monitoring-mass spectrometry (SRM-MS) |
| publicReleaseDate | 2016-07-04 00:00:00 |
| finalizedDate | 2016-07-04 02:59:23 |
| summary | Protein turnover represents an important mechanism in the functioning of cells, with deregulated synthesis and degradation of proteins implicated in many diseased states. Therefore, proteomics strategies to measure, with high confidence, turnover rates are of vital importance to understanding many biological processes. In this study, the more widely used approach of non-targeted precursor ion signal intensity (MS1) quantification is compared to selected reaction monitoring (SRM), a data acquisition strategy that records data for specific peptides, to determine if improved quantitative data would be obtained using a targeted quantification approach. Using mouse liver as a model system, turnover measurement of four tricarboxylic acid cycle proteins was performed using both MS1 and SRM quantification strategies. SRM outperformed MS1 in terms of sensitivity and selectivity of measurement, allowing more confident determination of protein turnover rates. SRM data is acquired using cheaper and more widely available tandem quadrupole mass spectrometers, making the approach accessible to a larger number of researchers than MS1 quantification, which is best performed on high mass resolution instruments. SRM acquisition is ideally suited to focussed studies where the turnover of tens of proteins is measured, making it applicable in determining the dynamics of proteins complexes and complete metabolic pathways. |
| contributors | Stephen W. Holman
Dean E. Hammond
Deborah M. Simpson
John Waters
Jane L. Hurst
Robert J. Beynon |
| publication | Holman, SW, Hammond, DE, Simpson, DM, Waters, J, Hurst, JL, and Beynon, RJ, Protein turnover measurement using selected reaction monitoring-mass spectrometry (SRM-MS), Philosophical Transactions of the Royal Society A, Accepted. |
| growth | Experimental animals
Eleven adult male C57BL/6JOlaHsd mice (Harlan UK Ltd., Shardlow, UK) of over 12 months of age at the start of the experiment were housed individually in 48 x 15 x 15 cm polypropylene cages (NKP Cages Ltd., Coalville, UK). Each cage contained substrate (Corn Cob Absorb 10-14 substrate), paper wool nest material and environmental enrichment. Food (LabDiet 5002 Certified Rodent Diet, Purina Mills, St. Louis, USA) and water were provided ad libitum. The mice were maintained on a reversed photo-period (light, 12 h; dark, 12 h; lights on at 20:00 hrs) and at 19-21 oC ± 2 oC. |
| treatment | Standard laboratory diet was replaced with a semisynthetic diet with the inclusion of [13C6]lysine at a relative isotope abundance (RIA) of 0.5. The dietary pellets were dissociated with water containing the dissolved [13C6]lysine to form a thick paste and mixed extensively. Once homogeneous, the paste was then extruded into strips 1 cm across and dried in a commercial foodstuff drying oven at 40 oC. The mice had access to the labelled diet for varying amounts of time: 0, 1, 2, 3, 4, 6, 9, 12, 17, 22 or 30 days exposure to the diet. The day that the animals were introduced to the labelled diet was staggered in order for all culls and dissections to take place on the same day. All mice were humanely killed on day 30 and dissected to recover the liver tissue from each animal. All resected tissue was frozen at -80 oC prior to analysis. |
| extraction | A small section of liver tissue was removed and further cut into small pieces prior to homogenisation in 1 mL of lysis buffer (7 M urea, 2 M thiourea, 2 % [w/v] CHAPS, 5 mM DTT) using a Precellys lysis kit (Stretton Scientific Ltd., Stretton, UK). Total protein extracted was quantified using a Bradford assay. |
| separation | One µg of peptides from each time-point were loaded in technical triplicates onto a trapping column (Symmetry C18, 180 µm x 2 cm, 5 µm packing material) in 99.9% A (0.1 % [v/v] formic acid):0.1 % B (0.1 % [v/v] formic acid in acetonitrile) for 3 min at a flow rate of 5 µL min-1. The trapping column was then set in-line with an analytical column (HSS T3 C18, 75 µm x 152 cm, 1.8 µm packing material) and the peptides eluted using a linear gradient of 97 % A:3 % B to 60 % A:40 % B over 60 min at a flow rate of 300 nL min-1, followed by washing at 95 % B for 5 min and reequilibration of the column to starting conditions. The column was maintained at 35 oC, and the effluent introduced into a nano-electrospray ionisation source operating in positive ion mode. |
| digestion | Two hundred µg quantities of protein were reduced, alkylated and digested with trypsin using a modified version of the filter-aided sample preparation (FASP) approach [38, 39]. |
| acquisition | Both quadrupoles were operated at unit mass resolution. Transitions were selected using a combination of in-house acquired data from the non-targeted DDA analysis, and publicly available peptide spectral libraries from NIST (www.peptide.nist.gov) for all peptides passing the criteria outlined above. This led to a total of between eight and 18 peptides being targeted for each protein. Between four and 13 product ions for each peptide were selected, and the samples analysed using an unscheduled SRM method to determine the two-to-four transitions with the highest signal-to-background ratios to monitor in the final quantitative assay. The peptides were separated into two categories based on their ionisation efficiency; “good” and “poor”. Methods were retention time scheduled into 3 min windows to achieve dwell times per transition of ≥ 16 ms for good ionisation efficiency peptides, and ≥ 28 ms for poor ionisation efficiency peptides. |
| informatics | Raw data was imported into Skyline, peak picking manually checked, peaks smoothed using Savitsky-Golay smoothing, and integrated peak areas exported for further processing. |
| instruments | Waters Xevo TQ-S |
| species | Mouse |
| massModifications | C+57.021464, K+6.020129 |
