On the relationship of protein and mRNA dynamics in vertebrate embryonic development
How the fertilized egg develops the anatomy of an adult ultimately must be explained at the biochemical level. Deeply connected to the anatomy is the complement of proteins and RNAs expressed during embryogenesis. The relationship of mRNA and respective protein expression dynamics during development has never been systematically explored. Here we present a comprehensive characterization of protein and mRNA dynamics across early development in Xenopus, between unfertilized egg and early tadpole stage. Surprisingly, most proteins change very little; there is an inverse correlation between abundance and rate of change. While the correlation of mRNA and protein expression is poor, a simple mass action kinetics model connects protein to RNA. Parameterized by two fixed parameters the median protein synthesis and degradation rates, the model depends on RNA dynamics, and initial protein concentration. Associated mRNA datasets in GEO: GSE73905 and GSE73870.
Sample Processing Protocol
Female Xenopus laevis were induced with 700U HCG. After 14 hours, eggs were washed with 1x MMR, and de-jellied with Cysteine (2% w/v), pH 8.0. Embryos of different stages were prepared from quantitative multiplexed analysis essentially as previously described ((Wühr et al., 2014), (Wühr et al., 2012) , Nuclear Proteome – under review). A pool of 20 embryos from stages 2, 7, 9, 12, 23, 33 were lysed with 1mL of 250 mM Sucrose, 1% NP40 Subtitute (Sigma), 5mM EDTA (pH 7.2), 1 Roche Complete mini tablet (EDTA free), 20 mM HEPES (pH 7.2), 10 μM Combretastatin 4A, and 10 μM Cyochalasin D. Lysate was pipetted up and down ten times with 1mL pipette, and vortexed for 10 seconds. Lysates were clarified by centrifugation at 10,000 rcf at 4 C for 4 minutes in a tabletop centrifuge. The cytoplasmic + lipid layers were mixed by gentle flicking and removed from the pelleted yolk. 450 µL of supernatatant was flash frozen with liquid nitrogen. After thawing SDS was added to to 2% (w/v). For cysteine protection, the sample was treated with 5 mM DTT for 20 minutes at 60°C, 15 mM NEM for 20 minutes at room temperature, and 5 mM DTT once more at room temperature. Proteins were isolated by methanol/chloroform precipitation (Wessel and Flügge, 1984). The protein pellet was resuspended in 100 µL of 9 M Urea, 50 mM HEPES (pH 8.5) and sonicated for five minutes. The sample was diluted to 4 M Urea with 50 mM HEPES (pH 8.5) and digested with LysC (Wako Chemicals) at 20 ng/μL at room temperature for 14 hours, then diluted with 50 mM HEPES (pH 8.5) to 2 M Urea and digested further with 40 ng/μL of LysC for 2 hours. From each condition ~200uL (~100ug of peptides) were labeled by addition of 0.2mg of TMT in 10uL of ACN. After 10minutes additional 10uL with 0.2mg of TMT were added. After one hour 0.5% w/v hydroxylamine was added to quench the reqction and incubated at room temperature for 30 minutes. The samples were pooled and after addition of 4% formic acid and speed vacing to remove ACN, the sample were pool and subjected to C18 solid phase extraction to isolate peptides (SPE) (SepPak, Waters). For fractionation the sample was separated by medium pH reverse-phase HPLC (Zorbax 300Extend- C18, 4.6 X 250 mm column, Agilant) in a 10 mM sodium carbonate buffer (pH 8.0) using an Acetonitrile gradient from 6% - 31%. With a flow rate of 0.8 mL/min, fractions were collected into a 96 well-plate every 38 seconds, then combined into two sets of twelve by pooling alternating wells from each column. Each fraction was dried and resuspened in 20 μL of 1% phosphoric acid. Peptides from each fraction were extracted with reverse-phase purification, (Lohse et al., 2012) resuspended in 1% formic acid and analyzed by LC-MS. LC-MS LC-MS experiments were performed on an Orbitrap Elite (Thermo Fischer Scientific). The Elite was equipped with a Famos autosampler (LC Packings) and an Agilent 1100 binary high pressure liquid chromatography (HPLC) pump (Agilent Technologies). For each run ~4 μg of peptides were separated on a 100 μm inner diameter microcapillary column packed first with approximately 0.5 cm of Magic C4 resin (5 µm, 200 Å, Michrom Bioresources) followed by 20 cm of Maccel C18 AQ resin (3 μm, 200 Å, Nest Group). Separation was achieved by applying a 5 - 27% acetonitrile gradient in 0.125% formic acid over 160 min at ~ 300 nl/min. Electrospray ionization was enabled through applying a voltage of 1.8 kV through a PEEK microtee at the inlet of the microcapillary column. The Elite was operated in data dependent mode. TMT quantification was performed with the MultiNotch MS3 method as previously described (McAlister et al., 2014). The survey scan was performed at a resolution setting of 70k (200m/z) followed by an MS2
Data Processing Protocol
MS data-analysis MS data-analysis was performed essentially as previously described (Nuclear proteome under review). A suite of in-house, developed software tools was used to convert mass spectrometric data from the RAW file to the mzXML format, as well as to correct erroneous assignments of peptide ion charge state and monoisotopic m/z (Huttlin et al., 2010). We used ReAdW.exe to convert the raw files into mzXML file format (http://sashimi.svn.sourceforge.net/viewvc/sashimi/). Assignment of MS2 spectra was performed using the SEQUEST algorithm (Eng et al., 1994) by searching the data against protein sequence database as described in the text attached with common contaminants like human keratins and trypsin. This forward database component was followed by a decoy component including all listed protein sequences in reversed order. Searches were performed using a 20 ppm precursor ion tolerance, where both peptide termini were required to be consistent with Trypsin or LysC specificity, while allowing one mis
Peshkin L, Wühr M, Pearl E, Haas W, Freeman RM Jr, Gerhart JC, Klein AM, Horb M, Gygi SP, Kirschner MW. On the Relationship of Protein and mRNA Dynamics in Vertebrate Embryonic Development. Dev Cell. 2015 Nov 9;35(3):383-94 PubMed: 26555057