Mass spectrometry identification and quantitation of proteins from small pools of developing auditory and vestibular cells
- Organism: Mouse
- Instrument: Q Exactive HF
Hair cells undergo postnatal development that leads to formation of their sensory organelles, synaptic machinery, and in the case of cochlear outer hair cells, their electromotile mechanism. To examine the proteome changes over development, we isolated pools of 5000 Pou4f3-Gfp positive or negative cells from the cochlea or utricles at three different developmental time points; these cell pools were analysed by data-dependent and data-independent acquisition (DDA and DIA) mass spectrometry. DDA data were used to generate spectral libraries, which enabled identification and accurate quantitation of specific proteins using the DIA datasets. We also isolated and pooled individual inner and outer hair cells from adult cochlea and compared their proteomes to those of developing hair cells.
Protein digests were separated using liquid chromatography with a NanoAcquity UPLC system (Waters); analytes were ionized using electrospray with a Nano Flex Ion Spray Source (Thermo Fisher) fitted with a 20 µm stainless steel nano-bore emitter spray tip and 1.0 kV source voltage, and were delivered to a Q Exactive HF (Thermo Fisher). Xcalibur version 4.0 was used to control the system. Samples were first bound to a trap cartridge (Symmetry C18 trap cartridge; Waters) at 15 µl/min to a for 10 min; the system then switched to a 75 µm x 250 mm NanoAcquity BEH 130 C18 column with 1.7 µm particles (Waters) using mobile phases water and acetonitrile containing 0.1% formic acid. A 7.5-30% acetonitrile gradient was delivered over 60 min at a flow rate of 300 nl/min. The DIA mass spectrometry method was constructed using 20 m/z quadrupole isolation windows. First, a full scan at 30,000 FWHM resolving power (at 200 m/z) was performed, followed by sequential HCD-MS/MS scans at normalized collision energy of 26 and 15,000 FWHM resolution. The range between 395 and 1005 m/z was surveyed by these experiments with maximum injection times of 55 ms and auto for MS and MS/MS, respectively. AGC values were set to 3 x 106 for MS and 1 x 106 for MS/MS. The MS/MS scan range was set to 200–2000 m/z. DIA data were analysed with Skyline23,24 version 3.7.
To selectively isolate hair cells, we used animals of either sex from the Tg(Pou4f3-Isl1-eGFP) transgenic mouse line, which expresses enhanced GFP under control of the Pou4f3 promoter. The high specificity of the Pou4f3 promoter ensures that the only labelled cells are hair cells. Utricles and cochleae were dissected in less than 1 hr using ice-cold PBS, then were transferred to ice-cold DMEM (Life Technologies) with 5% FBS. Cells were collected at three time points (postnatal day 0, 4 and 7) in triplicate. Cell aliquots were pooled and diluted if necessary to create samples of ~5000 GFP-positive or negative cells per condition. Cochlear hair cells are 0.5-1.0 pl in volume; if their total protein concentration was ~200 g/l, each cell would contain 0.1-0.2 ng of protein. Prior to sample preparation, the 5000 hair cells analysed in each sample thus together have 0.5-1.0 µg of total protein. The GFP-negative cells are likely somewhat larger and thus have larger amounts of total protein.
In-solution tryptic digests of the samples were prepared using an enhanced filter-aided sample preparation (eFASP) method. Proteins were digested in the filter unit in 100 µl digestion buffer with 200 ng sequencing-grade modified trypsin (Promega) at 37°C for 12-16 hours. Peptides were isolated by centrifugation and were extracted with ethyl acetate to remove remaining deoxycholic acid.
Created on 1/26/18 8:34 AM