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Trairak Pisitkun

Publications and source records attributed to Trairak Pisitkun.

13 recordsLinked to original sources
PubMed2026

Mass Spectrometry-Based Profiling of Personalized Immunopeptidomes in Thai Renal Cell Carcinoma.

This study profiles the personalized immunopeptidomes of 13 Thai patients with renal cell carcinoma (RCC), addressing a critical knowledge gap in Southeast Asian populations characterized by distinct HLA allele distributions. We combined whole-exome sequencing (WES)-based personalized proteome construction with liquid chromatography-tandem mass spectrometry (LC-MS/MS), using both database-driven searches and de novo peptide sequencing. HLA typing identified several class I allotypes that are underrepresented in publicly available immunopeptidome resources, including seven alleles not previously represented in the databases examined; HLA-A*11:01 was the most frequent allele in this cohort. Database-based analysis identified a single tumor-specific neoantigen derived from a mutant JADE2 peptide in the patient with the highest tumor mutational burden, which was validated by a mutant-specific ELISPOT response. In contrast, de novo sequencing revealed numerous noncanonical peptides, a subset of which were supported by proteogenomic validation using PepQuery and detected exclusively in cancer proteomes but not in normal tissue data sets, indicating their potential as tumor-associated antigen candidates. Together, these results establish an integrated and scalable framework for identifying HLA-presented tumor-derived peptides and provide a foundational immunopeptidome resource to support personalized cancer immunotherapy development in Southeast Asia.

Humans↗
PubMed2007

Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator.

Urinary exosomes are excreted from all nephron segments and may serve as biomarkers for classifying renal diseases. Isolation of urinary exosomes by the established ultracentrifugation method has some limitations for use in a clinical laboratory. We sought a rapid and simple way to obtain urinary exosomes. We used a commercially available nanomembrane concentrator to enrich exosomes from urine by centrifugation at 3,000 g for 10-30 min. Urinary exosomal markers tumor susceptibility gene 101, aquaporin-2, neuron-specific enolase, annexin V, angiotensin-converting enzyme, and podocalyxin (PODXL) were recovered from the nanomembrane concentrator and detected by Western blotting, and typical features of urinary vesicles were found by electron microscopy. Exosomal markers were detected in as little as 0.5 ml of urine. By the nanomembrane method, exosomal proteins could be recovered from urine samples frozen at -80 degrees C or refrigerated overnight at 4 degrees C then stored at -80 degrees C. By enriching exosomes we could detect PODXL, a podocyte marker, which decreased by 71% in five male patients with focal segmental glomerulosclerosis and abundant proteinuria. We conclude that 1) use of a nanomembrane concentrator simplifies and accelerates the enrichment of urinary exosomes; and 2) the nanomembrane concentrator can concentrate exosomal proteins from clinical urine samples. This enhanced method may accelerate the translation of urinary exosomal biomarkers from bench to bedside for the diagnosis, classification, and prognostication of renal diseases.

Biomarkers↗
PubMed2006

Dynamics of aquaporin-2 serine-261 phosphorylation in response to short-term vasopressin treatment in collecting duct.

We recently identified a novel phosphorylation site, serine-261 (pS261), in the COOH-terminus of the vasopressin-regulated water channel, aquaporin-2 (AQP2). To address whether phosphorylation at this site is regulated by vasopressin, a rabbit polyclonal phospho-specific antibody was generated. Dot blot and immunoblot analysis demonstrated that this antibody specifically recognizes AQP2 phosphorylated at pS261, and that phosphorylation of S256 (pS256), a site already known to be regulated by vasopressin, does not interfere with antibody recognition. Immunohistochemical analysis revealed intense pS261 labeling of inner medullary collecting duct (IMCD) from wild-type mice, while sections from AQP2 knockout animals showed a general absence of labeling. AQP2 pS261 was present in principal cells of all mouse and rat distal tubule segments from the connecting tubule to the terminal IMCD. Co-immunolabeling of collecting duct with phospho-specific and total AQP2 antibodies revealed that pS261 and pS256 have distinct subcellular distributions. Levels of pS256 increased, while the amount of pS261 significantly decreased in freshly isolated rat IMCD samples incubated with 1 nM [deamino-Cys(1),D-Arg(8)]vasopressin for 30 min. Similarly, based on immunohistochemical labeling, the amount of pS261 was reduced in all collecting duct segments of Brattleboro rats treated with [deamino-Cys(1),D-Arg(8)]vasopressin for 2 h. This study reveals a reciprocal change in S256 and S261 phosphorylation in response to short-term vasopressin exposure, suggesting that these residues may serve distinct roles in regulation of AQP2 subcellular distribution and collecting duct water permeability.

Amino Acid Sequence↗
PubMed2006

Automated quantification tool for high-throughput proteomics using stable isotope labeling and LC-MSn.

LC-MSn has become a popular option for high-throughput quantitative proteomics, thanks to the availability of stable-isotope labeling reagents. However, the vast quantity of data generated from LC-MSn continues to make the postacquisition quantification analyses challenging, especially in experiments involving multiple samples per experimental condition. To facilitate data analysis, we developed a computer program, QUIL, for automated protein quantification. QUIL accounts for the dynamic nature of spectral background and subtracts this background accordingly during ion chromatogram reconstruction. For elution profile identification, QUIL minimizes the inclusion of coeluted neighbor peaks, yet tolerates imperfect peak shapes. Outlier-resistant methods have been implemented for better protein ratio estimation. The utility of QUIL was validated by quantitative analyses of a standard protein as well as complex protein mixtures, which were labeled with cICAT or 18O and analyzed using LCQ, LTQ, or FT-ICR instruments. For samples that no prior knowledge of relative protein quantities was available, Western blotting was performed for confirmation. For the standard protein, the coefficient of variation (CV) of peptide ratio estimation was 6%. For complex mixtures, the median CV for protein ratio calculations was less than 10%. Computed protein abundance ratios exhibited a relatively high degree of correlation with those obtained from Western blot analyses. Compared with a widely used commercial software tool, QUIL showed improvement in ion chromatogram construction and peak integration and significantly reduced relative errors in abundance ratio assessment.

Animals↗
PubMed2006

LC-MS/MS analysis of apical and basolateral plasma membranes of rat renal collecting duct cells.

We used biotinylation and streptavidin affinity chromatography to label and enrich proteins from apical and basolateral membranes of rat kidney inner medullary collecting ducts (IMCDs) prior to LC-MS/MS protein identification. To enrich apical membrane proteins and bound peripheral membrane proteins, IMCDs were perfusion-labeled with primary amine-reactive biotinylation reagents at 2 degrees C using a double barreled pipette. The perfusion-biotinylated proteins and proteins bound to them were isolated with CaptAvidin-agarose beads, separated with SDS-PAGE, and sliced into continuous gel pieces for LC-MS/MS protein identification (LTQ, Thermo Electron Corp.). 17 integral and glycosylphosphatidylinositol (GPI)-linked membrane proteins and 44 non-integral membrane proteins were identified. Immunofluorescence confocal microscopy confirmed ACVRL1, H(+)/K(+)-ATPase alpha1, NHE2, and TauT expression in the IMCDs. Basement membrane and basolateral membrane proteins were biotinylated via incubation of IMCD suspensions with biotinylation reagents on ice. 23 integral and GPI-linked membrane proteins and 134 non-integral membrane proteins were identified. Analyses of non-integral membrane proteins preferentially identified in the perfusion-biotinylated and not in the incubation-biotinylated IMCDs revealed protein kinases, scaffold proteins, SNARE proteins, motor proteins, small GTP-binding proteins, and related proteins that may be involved in vasopressin-stimulated AQP2, UT-A1, and ENaC regulation. A World Wide Web-accessible database was constructed of 222 membrane proteins (integral and GPI-linked) from this study and prior studies.

Animals↗
PubMed2006

Discovery of urinary biomarkers.

A myriad of proteins and peptides can be identified in normal human urine. These are derived from a variety of sources including glomerular filtration of blood plasma, cell sloughing, apoptosis, proteolytic cleavage of cell surface glycosylphosphatidylinositol-linked proteins, and secretion of exosomes by epithelial cells. Mass spectrometry-based approaches to urinary protein and peptide profiling can, in principle, reveal changes in excretion rates of specific proteins/peptides that can have predictive value in the clinical arena, e.g. in the early diagnosis of disease, in classification of disease with regard to likely therapeutic responses, in assessment of prognosis, and in monitoring response to therapy. These approaches have potential value, not only in diseases of the kidney and urinary tract but also in systemic diseases that are associated with circulating small protein and peptide markers that can pass the glomerular filter. Most large scale biomarker discovery studies reported thus far have used one of two approaches to identify proteins and peptides whose excretion in urine changes in specific disease states: 1) two-dimensional electrophoresis with mass spectrometric and/or immunochemical identification of proteins and 2) top-down mass spectrometric methods (SELDI-TOF-MS and capillary electrophoresis-MS). These studies have been chiefly in the areas of nephrology, urology, and oncology. We review these applications, focusing on two areas of progress, viz. in bladder cancer and in acute rejection of renal transplants. Progress has been limited so far. However, with the advent of powerful LC-MS/MS methods along with methods for quantifying LC-MS/MS output, there is hope for an accelerated discovery and validation of disease biomarkers in urine.

Biomarkers↗
PubMed2006

Quantitative phosphoproteomics of vasopressin-sensitive renal cells: regulation of aquaporin-2 phosphorylation at two sites.

Protein phosphorylation plays a key role in vasopressin signaling in the renal-collecting duct. Large-scale identification and quantification of phosphorylation events triggered by vasopressin is desirable to gain a comprehensive systems-level understanding of this process. We carried out phosphoproteomic analysis of rat inner medullary collecting duct cells by using a combination of phosphopeptide enrichment by immobilized metal affinity chromatography and phosphorylation site identification by liquid chromatography-mass spectrometry(n) neutral loss scanning. A total of 714 phosphorylation sites on 223 unique phosphoproteins were identified from inner medullary collecting duct samples treated short-term with either calyculin A or vasopressin. A number of proteins involved in cytoskeletal reorganization, vesicle trafficking, and transcriptional regulation were identified. Previously unidentified phosphorylation sites were found for membrane proteins essential to collecting duct physiology, including eight sites among aquaporin-2 (AQP2), aquaporin-4, and urea transporter isoforms A1 and A3. Through label-free quantification of phosphopeptides, we identified a number of proteins that significantly changed phosphorylation state in response to short-term vasopressin treatment: AQP2, Bclaf1, LRRC47, Rgl3, and SAFB2. In the presence of vasopressin, AQP2 monophosphorylated at S256 and diphosphorylated AQP2 (pS256/261) increased in abundance, whereas AQP2 monophosphorylated at S261 decreased, raising the possibility that both sites are involved in vasopressin-dependent AQP2 trafficking. This study reveals the practicality of liquid chromatography-mass spectrometry(n) neutral loss scanning for large-scale identification and quantification of protein phosphorylation in the analysis of cell signaling in a native mammalian system.

Amino Acid Sequence↗
PubMed2006

High-throughput identification of IMCD proteins using LC-MS/MS.

The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based "IMCD Proteome Database" containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. Vasopressin analog (dDAVP) or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCDs were isolated for proteomic analysis. dDAVP and control samples were labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by one-dimensional SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification, based on semiquantitative immunoblotting of 16 proteins for which antibodies were available, showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and gamma-epithelial Na channel (gamma-ENaC), five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz., syntaxin-7, Rap1, GAPDH, heat shock protein (HSP)70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies.

Animals↗
PubMed2005

Large scale protein identification in intracellular aquaporin-2 vesicles from renal inner medullary collecting duct.

Vasopressin acts on renal collecting duct cells to stimulate translocation of aquaporin-2 (AQP2)-containing membrane vesicles from throughout the cytoplasm to the apical region. The vesicles fuse with the plasma membrane to increase water permeability. To identify the intracellular membrane compartments that contain AQP2, we carried out LC-MS/MS-based proteomic analysis of immunoisolated AQP2-containing intracellular vesicles from rat inner medullary collecting duct. Immunogold electron microscopy and immunoblotting confirmed heavy AQP2 labeling of immunoisolated vesicles. Vesicle proteins were separated by SDS-PAGE followed by in-gel trypsin digestion in consecutive gel slices and identification by LC-MS/MS. Identification of Rab GTPases 4, 5, 18, and 21 (associated with early endosomes); Rab7 (late endosomes); and Rab11 and Rab25 (recycling endosomes) indicate that a substantial fraction of intracellular AQP2 is present in endosomal compartments. In addition, several endosome-associated SNARE proteins were identified including syntaxin-7, syntaxin-12, syntaxin-13, Vti1a, vesicle-associated membrane protein 2, and vesicle-associated membrane protein 3. Rab3 was not found, however, either by mass spectrometry or immunoblotting, suggesting a relative lack of AQP2 in secretory vesicles. Additionally, we identified markers of the trans-Golgi network, components of the exocyst complex, and several motor proteins including myosin 1C, non-muscle myosins IIA and IIB, myosin VI, and myosin IXB. Beyond this, identification of multiple endoplasmic reticulum-resident proteins and ribosomal proteins indicated that a substantial fraction of intracellular AQP2 is present in rough endoplasmic reticulum. These results show that AQP2-containing vesicles are heterogeneous and that intracellular AQP2 resides chiefly in endosomes, trans-Golgi network, and rough endoplasmic reticulum.

Animals↗
PubMed2005

Prospects for urinary proteomics: exosomes as a source of urinary biomarkers.

Recent progress in biotechnology offers the promise of better medical care at lower costs. Among the techniques that show the greatest promise is mass spectrometry of proteins, which can identify proteins present in body fluids and tissue specimens at a large scale. Because urine can be collected in large amounts in a non-invasive fashion, the potential exists to use mass spectrometry to discover urinary biomarkers that are early predictors of renal disease, or useful in making therapeutic choices. Recently, the authors demonstrated that both membrane proteins and cytosolic proteins from renal epithelia are highly enriched in low-density urinary structures identified as exosomes. Exosomes were found to contain many disease-associated proteins including aquaporin-2, polycystin-1, podocin, non-muscle myosin II, angiotensin-converting enzyme, Na+ K+ 2Cl- cotransporter (NKCC2), thiazide-sensitive Na-Cl cotransporter (NCC), and epithelial sodium channel (ENaC). Potentially, other disease biomarkers could be discovered by mass spectrometry-based proteomic studies in well-defined patient populations. Herein is described the advantages of using urinary exosomes as a starting material for biomarker discovery. In addition, the purpose of this review is to present an overall strategy for biomarker discovery in urine using exosomes and for developing cost-effective clinical assays for these biomarkers, which can potentially be used for early detection of disease, as a means of differential diagnosis, or as a means of guiding therapy. Finally, potential barriers that need to be overcome before urinary proteomics can be applied clinically, are emphasized.

Biomarkers↗
PubMed2004

Identification and proteomic profiling of exosomes in human urine.

Urine provides an alternative to blood plasma as a potential source of disease biomarkers. One urinary biomarker already exploited in clinical studies is aquaporin-2. However, it remains a mystery how aquaporin-2 (an integral membrane protein) and other apical transporters are delivered to the urine. Here we address the hypothesis that these proteins reach the urine through the secretion of exosomes [membrane vesicles that originate as internal vesicles of multivesicular bodies (MVBs)]. Low-density urinary membrane vesicles from normal human subjects were isolated by differential centrifugation. ImmunoGold electron microscopy using antibodies directed to cytoplasmic or anticytoplasmic epitopes revealed that the vesicles are oriented "cytoplasmic-side inward," consistent with the unique orientation of exosomes. The vesicles were small (<100 nm), consistent with studies of MVBs and exosomes from other tissues. Proteomic analysis of urinary vesicles through nanospray liquid chromatography-tandem mass spectrometry identified numerous protein components of MVBs and of the endosomal pathway in general. Full liquid chromatography-tandem MS analysis revealed 295 proteins, including multiple protein products of genes already known to be responsible for renal and systemic diseases, including autosomal dominant polycystic kidney disease, Gitelman syndrome, Bartter syndrome, autosomal recessive syndrome of osteopetrosis with renal tubular acidosis, and familial renal hypomagnesemia. The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine.

Adult↗
PubMed2004

Comparison of different culture methods on bacterial recovery in hemodialysis fluids.

To examine the culture method that could provide the highest bacterial recovery, 143 reverse osmosis water samples used in hemodialysis were collected for comparison of the media (Tryptic Soy Agar, TSA vs Reasoner's 2A Agar, R2A), the temperature (20 degrees C vs 37 degrees C), the duration of incubation (48-hour vs 7-day), and the culture technique (membrane filtration vs spread plate methods). The European Best Practice Guideline method, R2A at 20 degrees Cfor 7-day incubation provided higher bacterial recovery than the Association for the Advancement of Medical Instrumentation (AAMI) method, TSA at 37 degrees C for 48-hour incubation. The membrane filtration method gave better yield than the spread plate method. As such, the European Best Practice Guideline method in combination with the membrane filtration technique would be the culture method of choice for hemodialysis fluids.

Agar↗