Mark R. Condina

Use of titanium dioxide to find phosphopeptide and total protein changes during epididymal sperm maturation

Although the overall performance of modern mass spectrometers has increased, proteomic analysis of complex samples still requires prefractionation either at the protein or peptide level to allow for in-depth analysis of normal cellular function. Here, we report a novel way to identify protein changes occurring during sperm development through the epididymis. Phosphopeptides were first enriched from either the rat caput or caudal regions of the epididymides using TiO(2), and the profiles then quantitatively compared. We show that 77 TiO(2)-enriched peptides become significantly modified in the epididymis, equating to 53 proteins. Through the use of immunoblot analysis, we confirmed that three proteins, ornithine-decarboxylase antizyme 3, heat-shock protein 90α, and testis-lipid binding protein, undergo major protein loss during epididymal passage. Many other proteins, including t-complex protein 10 and Spata18 show testis unique expression, appear to undergo phosphorylation during this same time frame. These data provide mechanistic insight into the means by which spermatozoa acquire functionality during epididymal transit.

Analysis of Phosphopeptide Changes as Spermatozoa Acquire Functional Competence in the Epididymis Demonstrates Changes in the Post-translational Modification of Izumo1

Spermatozoa are functionally inert when they emerge from the testes. Functional competence is conferred upon these cells during a post-testicular phase of sperm maturation in the epididymis. Remarkably, this functional transformation of epididymal spermatozoa occurs in the absence of nuclear gene transcription or protein translation. To understand the cellular mechanisms underpinning epididymal maturation, we have performed a label-free, MS-based, comparative quantification of peptides from caput, corpus and caudal epididymal spermatozoa. In total, 68 phosphopeptide changes could be detected during epididymal maturation corresponding to the identification of 22 modified proteins. Included in this list are the sodium-bicarbonate cotransporter, the sperm specific serine kinase 1, AKAP4 and protein kinase A regulatory subunit. Furthermore, four phosphopeptide changes came from Izumo1, the sperm-egg fusion protein, in the cytoplasmic segment of the protein. 2D-PAGE confirmed that Izumo1 is post-translationally modified during epididymal transit. Interestingly, phosphorylation on Izumo1 was detected on residue S339 in the caput and corpus but not caudal cells. Furthermore, Izumo1 exhibited four phosphorylated residues when spermatozoa reached the cauda, which were absent from caput cells. A model is advanced suggesting that these phospho-regulations are likely to act as a scaffold for the association of adaptor proteins with Izumo1 as these cells prepare for fertilization.

Cytokine receptor signaling activates an IKK-dependent phosphorylation of PUMA to prevent cell death

P53-upregulated modifier of apoptosis (PUMA), a pro-apoptotic member of the Bcl-2 family, is transcriptionally activated by p53 and is a key effector of p53-dependent apoptosis. We show that PUMA protein is subject to rapid post-translational regulation by phosphorylation at a conserved residue, serine 10, following serum or interleukin-3 (IL-3) stimulation. Serine 10 is not within the Bcl-2 homology (BH3) domain, and PUMA phosphorylated at serine 10 retained the ability to co-immunoprecipitate with antiapoptotic Bcl-2 family members. However, phosphorylated PUMA was targeted for proteasomal degradation indicating that it is less stable than unphosphorylated PUMA. Importantly, we identified IKK1/IKK2/Nemo as the kinase complex that interacts with and phosphorylates PUMA, thereby also demonstrating that IL-3 activates NFκB signaling. The identification and characterization of this novel survival pathway has important implications for IL-3 signaling and hematopoietic cell development.

A sensitive magnetic bead method for the detection and identification of tyrosine phosphorylation in proteins by MALDI-TOF/TOF MS

Phosphorylation is one of the most important PTMs and is estimated to occur on 30% of the mammalian proteome. Its perturbed regulation has been implicated in many pathologies. The rarity of phosphotyrosine compared with phosphoserine or phosphothreonine is prompting the development of more sensitive approaches because proteomic technologies that are currently used to assess tyrosine phosphorylation in proteins are inadequate, identifying only a fraction of the predicted tyrosine phosphoproteome. Here we describe the development of a reproducible, high‐sensitivity methodology for the detection and mapping of phosphotyrosine residues by MS. The anti‐phosphotyrosine antibody 4G10 was coupled covalently to super para‐magnetic beads or by affinity to super para‐magnetic beads with protein G covalently attached. Using this approach, we successfully enriched phosphotyrosine peptides mixed with non‐phosphorylated peptides at a ratio of up to 1:200, enabling detection at a level representing the highest sensitivity reported for tyrosine phosphorylation. The beads were subsequently used to enrich tyrosine phosphopeptides from a digest of the in vitro‐phosphorylated recombinant β‐intracellular region of the granulocyte‐macrophage colony‐stimulating factor receptor, which was subsequently analysed by MALDI‐TOF/TOF MS. Our results define this methodology as a sensitive approach for tyrosine phosphoproteome analysis.

The utility of isotope-coded protein labeling for prioritization of proteins found in ovarian cancer patient urine

Urine offers a number of attractive features as a sample type for biomarker discovery, including noninvasive sampling, quantity and availability, stability, and a narrow dynamic range. In this study we report the first application of isotope coded protein labeling (ICPL), coupled with in-solution isoelectric fractionation and LC-MALDI-TOF/TOF, to examine and prioritize urinary proteins from ovarian cancer patients. Following the definition of stringent exclusion criteria a total of 579 proteins were identified with 43% providing quantitation data. Protein abundance changes were validated for selected proteins by ESI-Qq-TOF MS, following which Western blot and immunohistochemical analysis by tissue microarray was used to explore the biological relevance of the proteins identified. Several established markers (e.g., HE4, osteopontin) were identified at increased levels in ovarian cancer patient urine, validating the approach used; we also identified a number of potential marker candidates (e.g., phosphatidylethanolamine binding protein 1, cell-adhesion molecule 1) previously unreported in the context of ovarian cancer. We conclude that the ICPL strategy for identification and relative quantitation of urine proteins is an appropriate tool for biomarker discovery studies, and can be applied for the selection of potential biomarker candidates for further characterization.

EZYprep LC-coupled MALDI-TOF/TOF MS: An improved matrix spray application for phosphopeptide characterisation

The quality of MALDI‐TOF mass spectrometric analysis is highly dependent on the matrix and its deposition strategy. Although different matrix‐deposition methods have specific advantages, one major problem in the field of proteomics, particularly with respect to quantitation, is reproducibility between users or laboratories. Compounding this is the varying crystal homogeneity of matrices depending on the deposition strategy used. Here, we describe a novel optimised matrix‐deposition strategy for LC‐MALDI‐TOF/TOF MS using an automated instrument that produces a nebulised matrix “mist” under controlled atmospheric conditions. Comparisons of this with previously reported strategies showed the method to be advantageous for the atypical matrix, 2,5‐DHB, and improved phosphopeptide ionisation when compared with deposition strategies for CHCA. This optimised DHB matrix‐deposition strategy with LC‐MALDI‐TOF/TOF MS, termed EZYprep LC, was subsequently optimised for phosphoproteome analysis and compared to LC‐ESI‐IT‐MS and a previously reported approach for phosphotyrosine identification and characterisation. These methods were used to map phosphorylation on epidermal growth factor‐stimulated epidermal growth factor receptor to gauge the sensitivity of the proposed method. EZYprep DHB LC‐MALDI‐TOF/TOF MS was able to identify more phosphopeptides and characterise more phosphorylation sites than the other two proteomic strategies, thus proving to be a sensitive approach for phosphoproteome analysis.

Mapping the testicular interstitial fluid proteome from normal rats.

Communication between the testicular somatic (Sertoli, Leydig, peritubular myoid, macrophage) and germ cell types is essential for sperm production (spermatogenesis), but the communicating factors are poorly understood. We reasoned that identification of proteins in the testicular interstitial fluid (TIF) that bathes these cells could provide a new means to explore spermatogenic function. The aim of this study was to map the proteome of TIF from normal adult rats. Low‐abundance proteins in TIF were enriched using ProteoMiner beads and identified by MALDI‐MS/MS, recognizing 276 proteins. Comparison with proteomic and genomic databases showed these proteins originated from germ cells, somatic cells (Sertoli, peritubular myoid, Leydig), and blood plasma. In silico analysis revealed homologues of >80% TIF proteins in the human plasma proteome, suggesting ready exchange between these fluids. Only 36% of TIF proteins were common with seminiferous tubule fluid that transports mature spermatids to the epididymis, indicating these two fluids are quite different. This TIF proteome provides an important new resource for the study of intercellular communication in the testis.

Quantitation and Identification of Intact Major Milk Proteins for High-Throughput LC-ESI-Q-TOF MS Analyses

Cow’s milk is an important source of proteins in human nutrition. On average, cow’s milk contains 3.5% protein. The most abundant proteins in bovine milk are caseins and some of the whey proteins, namely beta-lactoglobulin, alpha-lactalbumin, and serum albumin. A number of allelic variants and post-translationally modified forms of these proteins have been identified. Their occurrence varies with breed, individuality, stage of lactation, and health and nutritional status of the animal. It is therefore essential to have reliable methods of detection and quantitation of these proteins. Traditionally, major milk proteins are quantified using liquid chromatography (LC) and ultra violet detection method. However, as these protein variants co-elute to some degree, another dimension of separation is beneficial to accurately measure their amounts. Mass spectrometry (MS) offers such a tool. In this study, we tested several RP-HPLC and MS parameters to optimise the analysis of intact bovine proteins from milk. From our tests, we developed an optimum method that includes a 20-28-40% phase B gradient with 0.02% TFA in both mobile phases, at 0.2 mL/min flow rate, using 75°C for the C8 column temperature, scanning every 3 sec over a 600–3000 m/z window. The optimisations were performed using external standards commercially purchased for which ionisation efficiency, linearity of calibration, LOD, LOQ, sensitivity, selectivity, precision, reproducibility, and mass accuracy were demonstrated. From the MS analysis, we can use extracted ion chromatograms (EICs) of specific ion series of known proteins and integrate peaks at defined retention time (RT) window for quantitation purposes. This optimum quantitative method was successfully applied to two bulk milk samples from different breeds, Holstein-Friesian and Jersey, to assess differences in protein variant levels.

Tyrosine Phosphorylation Enrichment and Subsequent Analysis by MALDI‐TOF/TOF MS/MS and LC‐ESI‐IT‐MS/MS

This unit describes methods to detect, identify, and characterize tyrosine phosphorylation in proteins by mass spectrometry, including sample preparation methods, enrichment strategies using phosphotyrosine‐specific antibodies, and chromatographic separation methods. Curr. Protoc. Protein Sci. 62:13.11.1‐13.11.26.

Increased Phospho-Keratin 8 Isoforms in Colorectal Tumors Associated with EGFR Pathway Activation and Reduced Apoptosis

Hyperphosphorylated keratin (K) 8 acts as a phosphate “sponge” for stress-activated protein kinases thereby inhibiting pro-apoptotic molecules and thus apoptosis. MAP kinase/ERK1 has increased activity in colorectal cancer (CRC) and is known to phosphorylate K8. The aims were to identify the K8 isoforms abundantly present in colon tumors, using 2D difference gel electrophoresis (DIGE), to identify the modifications using mass spectrometry, and to validate the differential abundance of these isoforms in tumors relative to matched normal mucosae. 2D DIGE showed 3 isoforms of K8 significantly increased in tumor ≥2-fold in 6/8 pairs. Metal oxide affinity chromatography mass spectrometry and bioinformatics were used to identify phosphorylated serine residues. Levels of PS24, PS432, and PS74 by western blotting were found to be significantly increased in tumor versus matched normal. Blocking of EGFR signaling in Caco2 cells showed a significant decrease (P < 0.0001) in K8 PS74 and PS432 levels by 59% and 66%, respectively, resulting in increased apoptosis.