Human testis phosphoproteome reveals kinases as potential targets in spermatogenesis and testicular cancer
Spermatogenesis is a complex cell differentiation process that includes marked genetic, cellular functional and structural changes. It requires tight regulation, since disturbances in any of the spermatogenic stages would lead to fertility deficiencies. In order to increase our knowledge of signal transduction during sperm development, we carried out a large-scale identification of the phosphorylation events that occur in the human gonad. Metal oxide affinity chromatography using TiOx combined with LC-MS/MS was conducted to profile the phosphoproteome of human testes with full spermatogenesis. A total of 8187 phosphopeptides derived from 2661 proteins were identified, resulting in the most complete report of human testicular phosphoproteins to date. Phosphorylation events were enriched in proteins functionally related to spermatogenesis, as well as to highly active processes in the male gonad, such as transcriptional and translational regulation, cytoskeleton organization, DNA packaging, cell cycle and apoptosis. Moreover, 174 phosphorylated kinases were identified. The most active and abundant human protein kinases in the testis were predicted both by the phosphorylation status of the kinase activation loop and the number of phosphopeptide spectra identified. The potential function of two of those kinases, cyclin-dependent kinase 12 (CDK12) and p21-activated kinase 4(PAK4), has been explored by protein-protein interaction analysis, immunodetection in human and mouse testicular tissue, and functional assay in a human embryonal carcinoma cell line. The co-localization of CDK12 with Golgi markers and probably pro-acrosomal vesicles suggests a potential crucial role of this protein kinase in sperm formation. PAK4 expression has been found limited to human spermatogonia, and a role in embryonal carcinoma cell response to apoptosis has been observed. Together, our data confirm that phosphoregulation by protein kinases is highly active in sperm differentiation, and open a window to detailed characterization and validation of potential targets for the development of drugs modulating male fertility, and tumor behavior.
Sample Processing Protocol
Testicular tissue from three individuals was lysed in lysis buffer containing 9 M Urea, 20 mM HEPES pH 8.0, 1 mM Na3VO4 (orhovanadate), 2.5 mM Na4P2O7 (pyrophosphate), and 1 mM Na2C3H7PO6 (β-Glycerophosphate), by vortexing and sonication. After lysis, protein concentration was determined using the BCA method (ThermoPierce, Rockford, IL, US). Tissue lysates were reduced in 4.5 mM DTT for 30 min at 55 °C, cooled to room temperature, and alkylated in 11 mM iodoacetamide for 15 min in the dark. Subsequently, tissue lysates were diluted in 20 mM HEPES pH 8.0 to reduce the urea concentration to 2 M, and digested overnight with trypsin (Promega, Madison, WI, US) with an enzyme:protein ratio of 1:50 (w/w). Phosphopeptide enrichment. For each testis tissue sample, 500 µg of tryptic lysate digests were acidified by adding TFA to a final concentration of 1%, and incubated on ice for 15 min. Samples were desalted with 30 mg OASIS HLB cartridges (Waters Corporation, Milford, MA, US), previously activated in 100% ACN and equilibrated in 0.1% TFA. Briefly, peptides were loaded in the cartridge, washed in 0.1% TFA, and eluted in 0.1% TFA/80% ACN solution. Subsequently, desalted peptides were diluted 1:1 with lactic acid solution (0.3 g/ml lactic acid, 0.07% TFA, 53% ACN). For TiOx capture, 2.5 mg of TiO2 beads (GL sciences, 10 µm) were packed in a 200-µl pipette tip fitted with a 16G-needle punch of C8 material at the narrow end. Tips containing the TiOx bed were washed with 200 µl of 0.1% TFA/80% ACN and equilibrated with 200 µl of 300 mM acid lactic solution. Desalted peptides were loaded in the tips in 5 cycles of 200 µl of peptide mixture and centrifuged at 1500 G for 4 min. The TiOx bed with bound phosphopeptides was then washed, firstly with 200 µl lactic acid solution, and secondly with 200 µl 0.1% TFA/80% ACN. All steps were performed by centrifugation at 1500 G for 4 min. Phosphopeptides were eluted in two steps with 50 µl 0.5% piperidine (Thermo Fisher Scientific) and 50 µl 5% piperidine, and subsequently quenched in 100 µl 20% H3PO4. Phosphopeptides were desalted using 200-µl pipette tips fitted with a 16G-needle punch of SDB-XC SPE material at the narrow end, which was previously washed with 20 µl 0.1% TFA/80% ACN and equilibrated with 20 µl 0.1% TFA. Phosphopeptides were loaded and centrifuged for 3 min at 1000G. SDB-XC beds were then washed with 20 µl of 0.1% TFA, and desalted phosphopeptides were eluted with 20 µl of 0.1% TFA/80% ACN. Phosphopeptides were dried in a vacuum centrifuge and dissolved in 20 µl 0.5% TFA/4% ACN prior to injection. LC-MS/MS Peptides were separated by an Ultimate 3000 nanoLC-MS/MS system (Dionex LC-Packings, Amsterdam, The Netherlands) equipped with a 20 cm × 75 μm ID fused silica column custom packed with 1.9 μm 120 Å ReproSil Pur C18 aqua (Dr Maisch GMBH, Ammerbuch-Entringen, Germany). After injection, peptides were trapped at 6 μl/min on a 10 mm × 100 μm ID trap column packed with 5 μm 120 Å ReproSil Pur C18 aqua at 2% buffer B (buffer A: 0.5% acetic acid (Fischer Scientific), buffer B: 80% ACN, 0.5% acetic acid) and separated at 300 nl/min in a 10–40% buffer B gradient in 90 min (120 min inject-to-inject). Eluting peptides were ionized at a potential of +2 kVa into a Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Intact masses were measured at resolution 70.000 (at m/z 200) in the orbitrap using an AGC target value of 3E6 charges. The top 10 peptide signals (charge-states 2+ and higher) were submitted to MS/MS in the HCD (higher-energy collision) cell (1.6 amu isolation width, 25% normalized collision energy). MS/MS spectra were acquired at resolution 17.500 (at m/z 200) in the orbitrap using an AGC target value of 2E5 charges and an underfill ratio of 0.1%. Dynamic exclusion was applied with a repeat count of 1 and an exclusion time of 30 s.
Data Processing Protocol
Peptide and Protein identification MS/MS spectra were searched against Swissprot human proteome (canonical and isoforms, release 2015_09, 42122 entries) using MaxQuant 188.8.131.52. Enzyme specificity was set to trypsin and up to two missed cleavages were allowed. Cysteine carboxamidomethylation (Cys, +57.021464 Da) was treated as fixed modification and serine, threonine and tyrosine phosphorylation (+79.966330 Da), methionine oxidation (Met,+15.994915 Da) and N-terminal acetylation (N-terminal, +42.010565 Da) as variable modifications. Peptide precursor ions were searched with a maximum mass deviation of 4.5 ppm and fragment ions with a maximum mass deviation of 20 ppm. Peptide, protein and site identifications were filtered at an FDR of 1% using the decoy database strategy. The minimal peptide length was 7 amino-acids and the minimum Andromeda score for modified peptides was 40 and the corresponding minimum delta score was 6 (default MaxQuant settings). Peptide identifications were propagated across samples using the match between runs (MBR) option checked. Lysates were searched with the same parameters but without phosphorylation as variable modification and MBR unchecked. Label-free phosphopeptide quantification Phosphopeptides were quantified by counting MS/MS spectra (spectral counts) or by their extracted ion intensities (‘Intensity’ in MaxQuant). For each sample the phosphopeptide intensities were normalized on the median intensity of all identified proteins (from the lysate MaxQuant Evidence table) in the lysate of each corresponding sample (‘normalised intensity’).
Sander Piersma, OncoProteomics Laboratory, dept of Medical Oncology, VUmc Medical Center, Amsterdam, The Netherlands
Connie Ramona Jimenez, OncoProteomics Laboratory, Dept of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands ( lab head )
Castillo J, Knol JC, Korver CM, Piersma SR, Pham TV, Goeij de Haas RR, van Pelt AMM, Jimenez CR, Jansen BJH. Human testis phosphoproteome reveals kinases as potential targets in spermatogenesis and testicular cancer. Mol Cell Proteomics. 2019 PubMed: 30683686