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Immunoaffinity targeted mass spectrometry analysis of plasma samples reveals an imbalance of active and inactive CXCL10 in primary Sjögren’s Syndrome disease patients

ABSTRACT: One of the most important advantages of mass spectrometry is the ability to quantify proteins and their modifications in parallel to obtain a holistic picture of the protein of interest. Here, we present a hybrid immunoaffinity targeted mass spectrometry (MS) approach that combines efficient pan-antibody enrichment of a specific protein from plasma with the selectivity of targeted MS analysis to quantitate specific protein modifications. In this study, we used this approach to quantify plasma levels of the chemokine CXCL10 that has been associated with many immunological disorders such as systemic lupus erythematosus and primary Sjögren's Syndrome. The hybrid approach enabled sensitive, specific and simultaneous quantification of total, full-length (active) CXCL101-77 and DPP4 truncated (inactive) CXCL103-77 in human plasma. Samples from 30 healthy individuals and 34 primary Sjögren's Syndrome patients were analyzed. The ratio of CXCL101-77 to truncated CXCL103-77 was significantly increased and demonstrated an improved classification of the primary Sjögren's syndrome patients (ROC AUC = 0.74) when compared to total CXCL10 (ROC AUC = 0.66). Furthermore, the ratio of CXCL101-77 to truncated CXCL103-77 correlated best with Sjögren's syndrome disease activity. As this strategy can be readily adapted to other proteins and modifications of interest, we are convinced that it will lead to a more detailed understanding of different proteoforms in physiology and pathology yielding more relevant biomarkers and drug targets.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood

SUBMITTER: Alexander Schmidt

LAB HEAD: ALexander Schmidt

PROVIDER: PXD019709 | Pride | 2020-08-20

REPOSITORIES: Pride

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Project description:Background: Circular RNA (circRNA) has been reported to be involved in the pathogenesis of cardiovascular disease (CVD), however, it is unclear whether circRNA carried by exosomes (exos) can be used as biomarkers for chronic coronary syndrome (CCS). This study aimed to investigate the expression pattern of exosomal circRNAs in the plasma of CCS patients, and to screen exo-circRNAs that can act as biomarkers for the diagnosis of CCS. Methods: High-throughput sequencing technology was carried out in the plasma exosomal RNA of 15 CCS patients and 15 non-cardiac chest pain patients (NCCP, control group) (sequencing cohort) to screen for differentially expressed circRNAs. Selected differentially expressed exo-circRNAs were further verified by real time polymerase chain reaction (RT-PCR) in a small-sample cohort (derivation cohort) and a large-sample cohort (validation cohort). Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic value of exo-circRNAs for CCS patients. Results: By high-throughput sequencing of circRNAs in 15 CCS patients and 15 NCCP patients, 276 circRNAs differentially expressed in plasma exosomes of CCS patients were screened by using |log2(Fold change) |>1 and q-value<0.001, and 103 were up-regulated and 173 were down-regulated. Among the 103 up-regulated circRNAs, 5 circRNAs with high expression levels and significantly increased in plasma exosomes of CCS patients were selected for validation. Twice RT-PCR validation demonstrated that exo-hsa_circ_0075269 and exo-hsa_circ_0000284 were significantly up-regulated in patients with CCS (P<0.001). Circulating exo-hsa_circ_0075269 and exo-hsa_circ_0000284 yielded the area under the curve (AUC) values of 0.761 (P<0.001, 95%CI=0.669, 0.852) and 0.623 (P=0.015, 95%CI=0.522, 0.724) for CCS, respectively by ROC curve analysis. Moreover, AUC of hsa_circ_0075269 combined with hsa_circ_0000284 for diagnosing CCS was 0.741 (P<0.001, 95%CI=0.667, 0.850). Conclusion: The expression profile of circRNA in plasma exosomes of patients with CCS was significantly different from that of the control group. Plasma exo-hsa_circ_0075269 and exo-hsa_circ_0000284 have the potential to be new biomarkers for CCS diagnosis.

Project description:Background Testosterone (T) is the main androgen playing a crucial role in regulating the function of both reproductive and non-reproductive organs. Current methods for assessing T levels in humans do not reflect the biological androgenic activity (BAA), which may be important for example in the diagnosis of hypogonadism and clarifying pathogenetic mechanisms linking androgen deficiency to various comorbidities. In this study, we aimed to identify new protein markers of BAA in humans and test their predictive value in relation to some androgen deficiency linked pathologies. Methods and Findings Plasma samples (n=90) from 30 healthy chemically castrated young volunteer males (19-32 years) were collected at three different time points: baseline – prior to treatment with GnRH antagonist), three weeks after GnRH antagonist treatment, and at the end of the study, two weeks thereafter. Following the second blood sampling, all men were supplemented with 1000 mg T undecanoate. The samples were analysed by mass spectrometry-based proteomics. A total of 676 proteins were identified among which expression of 46 was statistically significantly associated with T concentration (p<0.05, ANOVA paired). Based on ROC-analysis, 3 candidate proteins were selected, 4-hydroxyphenylpyruvate dioxygenase (4HPPD), insulin-like growth factor-binding protein 6 (IGFBP6) and fructose-bisphosphate aldolase (ALDOB). Together with a mathematically calculated variable (MMA: Multi-Marker Algorithm), based on the combination of 4HPPD and IGFBP6 values, their predictive value in relation to low T levels and cardio-metabolic risk factors was tested in 75 males suffering from infertility, 32-43 years of age. Of these males, four were diagnosed with type 2-diabetes and 14 with the metabolic syndrome. Clinical chemistry data regarding cardio-metabolic parameters, bone mineral density and androgen receptor CAG repeat lengths were assessed. The three proteins significantly distinguished between low (< 8 nmol/L) and normal (> 12 nmol/L) morning fasting T (AUC: 0.70-0.77). The MMA had an even higher accuracy (AUC=0.80) in identifying subjects with low T. 4HPPD, ALDOB and MMA showed higher accuracy than T (AUC=0.85-0.92 vs. AUC=0.55; p<.001) in identifying infertile patients with type 2 diabetes mellitus and metabolic syndrome (AUC=0.74-0.78 vs. AUC=0.56; p<0.05). These markers also showed higher AUC than T in predicting adverse cardiovascular risk lipid profile and insulin resistance patients but the differences were not statistically significant. 4HPPD and MMA distinguished patients with low bone density (z-score in DEXA scan < -1), but no difference was observed when comparing with AUC for T,. The androgenic dependence of 4HPPD and ALDOB were confirmed by finding statistically significant lower levels of these markers in men with androgen CAG repeat lengths < 21 (p=0.002) and >22 (p=0.038) as compared to those with CAG of 21-22. No statistical significances were found for IGFBP6. Conclusions 4HPPD and ALDOB were identified as potential biomarkers for diagnosis of male hypogonadism and in identifying subjects at high risk of some of its clinical sequelae. More extensive testing should be carried out in order to elucidate their usefulness in clinical assessment of androgenic activity, not only in adult males but also in women and in pre-pubertal boys.

			Action	DRS
	001_1-16r_C17_CHECK.raw	Raw
	001_DIL_PRM_C19.raw	Raw
	001_DIL_PRMr_C19.raw	Raw
	001_trunc_D18.raw	Raw
	001_trunc_D18F053377.mzid.gz	Mzid

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Immunoaffinity targeted mass spectrometry analysis of plasma samples reveals an imbalance of active and inactive CXCL10 in primary Sjögren’s Syndrome disease patients

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