Variable blood processing procedures contribute to plasma proteomic variability

Abstract Background Plasma is a potentially rich source of protein biomarkers for disease progression and drug response. Large multi-center studies are often carried out to increase the number of samples analyzed in a given study. This may increase the chances of variation in blood processing and handling, leading to altered proteomic results. This study evaluates the impact of blood processing variation on LC–MS/MS proteomic analysis of plasma. Methods Initially two batches of patient plasma samples (120 and 204 samples, respectively) were analyzed using LC–MS/MS shotgun proteomics. Follow-up experiments were designed and carried out on healthy donor blood in order to examine the effects of different centrifugation conditions, length of delay until first centrifugation, storage temperature and anticoagulant type on results from shotgun proteomics. Results Variable levels of intracellular proteins were observed in subsets of patient plasma samples from the initial batches analyzed. This observation correlated strongly with the site of collection, implicating variability in blood processing procedures. Results from the healthy donor blood analysis did not demonstrate a significant impact of centrifugation conditions to plasma proteome variation. The time delay until first centrifugation had a major impact on variability, while storage temperature and anticoagulant showed less pronounced but still significant effects. The intracellular proteins associated with study site effect in patient plasma samples were significantly altered by delayed processing also. Conclusions Variable blood processing procedures contribute significantly to plasma proteomic variation and may give rise to increased intracellular proteins in plasma. Accounting for these effects can be important both at study design and data analysis stages. This understanding will be valuable to incorporate in the planning of protein-based biomarker discovery efforts in the future.

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Human Plasma Metabolomics for Biomarker Discovery: Targeting the Molecular Subtypes in Breast Cancer

Cancers ◽

10.3390/cancers13010147 ◽

2021 ◽

Vol 13 (1) ◽

pp. 147

Author(s):

Leticia Díaz-Beltrán ◽

Carmen González-Olmedo ◽

Natalia Luque-Caro ◽

Caridad Díaz ◽

Ariadna Martín-Blázquez ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Biomarker Discovery ◽

Molecular Subtypes ◽

Breast Cancer Subtype ◽

P Value ◽

Plasma Samples ◽

Breast Cancer Patients ◽

Clinical Value ◽

Molecular Phenotypes

Purpose: The aim of this study is to identify differential metabolomic signatures in plasma samples of distinct subtypes of breast cancer patients that could be used in clinical practice as diagnostic biomarkers for these molecular phenotypes and to provide a more individualized and accurate therapeutic procedure. Methods: Untargeted LC-HRMS metabolomics approach in positive and negative electrospray ionization mode was used to analyze plasma samples from LA, LB, HER2+ and TN breast cancer patients and healthy controls in order to determine specific metabolomic profiles through univariate and multivariate statistical data analysis. Results: We tentatively identified altered metabolites displaying concentration variations among the four breast cancer molecular subtypes. We found a biomarker panel of 5 candidates in LA, 7 in LB, 5 in HER2 and 3 in TN that were able to discriminate each breast cancer subtype with a false discovery range corrected p-value < 0.05 and a fold-change cutoff value > 1.3. The model clinical value was evaluated with the AUROC, providing diagnostic capacities above 0.85. Conclusion: Our study identifies metabolic profiling differences in molecular phenotypes of breast cancer. This may represent a key step towards therapy improvement in personalized medicine and prioritization of tailored therapeutic intervention strategies.

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Proteomics in Diagnosis of Prostate Cancer/ Протеомика Во Дијагноза На Простатниот Карцином

PRILOZI ◽

10.1515/prilozi-2015-0027 ◽

2015 ◽

Vol 36 (1) ◽

pp. 5-36 ◽

Cited By ~ 3

Author(s):

Katarina Davalieva ◽

Momir Polenakovic

Keyword(s):

Prostate Cancer ◽

Tumor Tissue ◽

Biomarker Discovery ◽

Molecular Level ◽

Specific Antigen ◽

Shotgun Proteomics ◽

Comparative Proteomics ◽

Label Free ◽

The Past ◽

Proteomics Research

Abstract Prostate cancer (PCa) is the second most frequently diagnosed malignancy in men worldwide. The introduction of prostate specific antigen (PSA) has greatly increased the number of men diagnosed with PCa but at the same time, as a result of the low specificity, led to overdiagnosis, resulting to unnecessary biopsies and high medical cost treatments. The primary goal in PCa research today is to find a biomarker or biomarker set for clear and effecttive diagnosis of PCa as well as for distinction between aggressive and indolent cancers. Different proteomic technologies such as 2-D PAGE, 2-D DIGE, MALDI MS profiling, shotgun proteomics with label-based (ICAT, iTRAQ) and label-free (SWATH) quantification, MudPIT, CE-MS have been applied to the study of PCa in the past 15 years. Various biological samples, including tumor tissue, serum, plasma, urine, seminal plasma, prostatic secretions and prostatic-derived exosomes were analyzed with the aim of identifying diagnostic and prognostic biomarkers and developing a deeper understanding of the disease at the molecular level. This review is focused on the overall analysis of expression proteomics studies in the PCa field investigating all types of human samples in the search for diagnostics biomarkers. Emphasis is given on proteomics platforms used in biomarker discovery and characterization, explored sources for PCa biomarkers, proposed candidate biomarkers by comparative proteomics studies and the possible future clinical application of those candidate biomarkers in PCa screening and diagnosis. In addition, we review the specificity of the putative markers and existing challenges in the proteomics research of PCa.

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Transcriptome and Proteome Research in Veterinary Science: What Is Possible and What Questions Can Be Asked?

The Scientific World JOURNAL ◽

10.1100/2012/254962 ◽

2012 ◽

Vol 2012 ◽

pp. 1-14 ◽

Cited By ~ 12

Author(s):

Robert Klopfleisch ◽

Achim D. Gruber

Keyword(s):

Sequential Analysis ◽

Biomarker Discovery ◽

Expression Patterns ◽

Proteome Analysis ◽

Shotgun Proteomics ◽

Complete Analysis ◽

Biological Research ◽

Veterinary Research ◽

Gene Sets ◽

2D Gel

In recent years several technologies for the complete analysis of the transcriptome and proteome have reached a technological level which allows their routine application as scientific tools. The principle of these methods is the identification and quantification of up to ten thousands of RNA and proteins species in a tissue, in contrast to the sequential analysis of conventional methods such as PCR and Western blotting. Due to their technical progress transcriptome and proteome analyses are becoming increasingly relevant in all fields of biological research. They are mainly used for the explorative identification of disease associated complex gene expression patterns and thereby set the stage for hypothesis-driven studies. This review gives an overview on the methods currently available for transcriptome analysis, that is, microarrays, Ref-Seq, quantitative PCR arrays and discusses their potentials and limitations. Second, the most powerful current approaches to proteome analysis are introduced, that is, 2D-gel electrophoresis, shotgun proteomics, MudPIT and the diverse technological concepts are reviewed. Finally, experimental strategies for biomarker discovery, experimental settings for the identification of prognostic gene sets and explorative versus hypothesis driven approaches for the elucidation of diseases associated genes and molecular pathways are described and their potential for studies in veterinary research is highlighted.

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Does Ramadan Fasting Affect Hydration Status and Kidney Function in CKD Patients?

Annals of Nutrition and Metabolism ◽

10.1159/000486799 ◽

2018 ◽

Vol 72 (3) ◽

pp. 241-247 ◽

Cited By ~ 9

Author(s):

Shadia Hassan ◽

Fadi Hassan ◽

Nur Abbas ◽

Kamal Hassan ◽

Nihal Khatib ◽

...

Keyword(s):

Kidney Function ◽

Estimated Glomerular Filtration Rate ◽

Blood Analysis ◽

Hydration Status ◽

Control Group ◽

Significant Deterioration ◽

Ramadan Fasting ◽

Reasonable Degree ◽

Fast Control ◽

The Impact

Background/Aims: This study is the first of its kind to examine the impact of the Ramadan fasting on hydration status, plasma brain natriuretic peptide (BNP) levels, and kidney function in chronic kidney disease (CKD) patient. Methods: This prospective cohort study included 2 groups of patients with CKD grades 2–4: thirty-one Muslim patients who fasted the month of Ramadan (fasting group) and 26 Muslim patients who did not fast (control group). One week before the Ramadan fast, in the last week of the month of Ramadan (4 weeks), and 4 weeks after the end of the Ramadan month (8 weeks), hydration status and blood analysis of urea, creatinine and BNP levels were measured. Results: Among fasting patients, serum urea levels increased significantly (p = 0.024) during the last week of fasting and returned to basal levels at 4 weeks after the end of the Ramadan month, the estimated glomerular filtration rate did not change significantly at the end of fasting (p = 0.411), the hydration status indices and plasma BNP levels were significantly decreased after fasting (p ≤ 0.021) but returned to basal values 4 weeks thereafter. Conclusions: Patients with CKD grades 2–4 can fast throughout the month of Ramadan with no significant deterioration of renal functions and with a reasonable degree of safety.

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The impact of blood-processing time on the proteome of human peripheral blood mononuclear cells

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics ◽

10.1016/j.bbapap.2020.140581 ◽

2021 ◽

Vol 1869 (3) ◽

pp. 140581

Author(s):

Bernardo Bonilauri ◽

Marlon D.M. Santos ◽

Amanda Caroline Camillo-Andrade ◽

Saloê Bispo ◽

Fabio C.S. Nogueira ◽

...

Keyword(s):

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Processing Time ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Peripheral Blood Mononuclear ◽

Blood Processing ◽

Blood Mononuclear Cells ◽

The Impact

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Effect of Equilibrated pH and Indigenous Spoilage Microorganisms on the Inhibition of Proteolytic Clostridium botulinum Toxin Production in Experimental Meals under Temperature Abuse

Journal of Food Protection ◽

10.4315/0362-028x.jfp-17-012 ◽

2017 ◽

Vol 80 (8) ◽

pp. 1252-1258 ◽

Cited By ~ 2

Author(s):

Max C. Golden ◽

Brandon J. Wanless ◽

Jairus R. D. David ◽

D. Scott Lineback ◽

Ryan J. Talley ◽

...

Keyword(s):

Botulinum Toxin ◽

Clostridium Botulinum ◽

Storage Temperature ◽

Phase 1 ◽

Toxin Production ◽

Brussels Sprouts ◽

The Impact ◽

And Storage ◽

Spoilage Microorganisms ◽

Toxin Formation

ABSTRACT Clostridium botulinum is a foreseeable biological hazard in prepared refrigerated meals that needs to be addressed in food safety plans. The objective of this study was to evaluate the effect of product composition and storage temperature on the inhibition of botulinum toxin formation in nine experimental meals (meat, vegetable, or carbohydrate based). Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin in samples stored at 25°C for up to 96 h for phase 1, or at 25°C for 12 h and then transferred to 12.5°C for up to 12 and 6 weeks in phases 1 and 2, respectively. For phase 1, none of the treatments (equilibrated pH 5.8) supported toxin production when stored at 25°C for 48 h, but toxin production was observed in all treatments at 72 h. For the remaining experiments with storage at 12.5°C, toxin production was dependent on equilibrated pH, storage time, and growth of indigenous spoilage microorganisms. In phase 1, no gross spoilage and no botulinum toxin was detected for any treatment (pH ≤5.8) stored at 12.5°C for 12 weeks. In phase 2, gross spoilage varied by commodity, with the brussels sprouts meal with pH 6.5 showing the most rapid spoilage within 2 weeks and botulinum toxin detected at 5 and 6 weeks for the control and cultured celery juice treatments, respectively. In contrast, spoilage microbes decreased the pH of a pH 5.9 beef treatment by 1.0 unit, potentially inhibiting C. botulinum through 6 weeks at 12.5°C. None of the other treatments with pH 5.8 or below supported toxin production or spoilage. This study provides validation for preventive controls in refrigerated meals. These include equilibrated product pH and storage temperature and time to inhibit toxin formation by proteolytic C. botulinum, but the impact of indigenous microflora on safety and interpretation of challenge studies is also highlighted.

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Gel-Based Proteomics of Clinical Samples Identifies Potential Serological Biomarkers for Early Detection of Colorectal Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms20236082 ◽

2019 ◽

Vol 20 (23) ◽

pp. 6082 ◽

Cited By ~ 3

Author(s):

Stine Thorsen ◽

Irina Gromova ◽

Ib Christensen ◽

Simon Fredriksson ◽

Claus Andersen ◽

...

Keyword(s):

Colorectal Cancer ◽

Early Detection ◽

Biomarker Discovery ◽

Real Life ◽

Clinical Samples ◽

Plasma Samples ◽

Healthy Individuals ◽

Protein Biomarkers ◽

2D Gel ◽

Cancer Diseases

The burden of colorectal cancer (CRC) is considerable—approximately 1.8 million people are diagnosed each year with CRC and of these about half will succumb to the disease. In the case of CRC, there is strong evidence that an early diagnosis leads to a better prognosis, with metastatic CRC having a 5-year survival that is only slightly greater than 10% compared with up to 90% for stage I CRC. Clearly, biomarkers for the early detection of CRC would have a major clinical impact. We implemented a coherent gel-based proteomics biomarker discovery platform for the identification of clinically useful biomarkers for the early detection of CRC. Potential protein biomarkers were identified by a 2D gel-based analysis of a cohort composed of 128 CRC and site-matched normal tissue biopsies. Potential biomarkers were prioritized and assays to quantitatively measure plasma expression of the candidate biomarkers were developed. Those biomarkers that fulfilled the preset criteria for technical validity were validated in a case-control set of plasma samples, including 70 patients with CRC, adenomas, or non-cancer diseases and healthy individuals in each group. We identified 63 consistently upregulated polypeptides (factor of four-fold or more) in our proteomics analysis. We selected 10 out of these 63 upregulated polypeptides, and established assays to measure the concentration of each one of the ten biomarkers in plasma samples. Biomarker levels were analyzed in plasma samples from healthy individuals, individuals with adenomas, CRC patients, and patients with non-cancer diseases and we identified one protein, tropomyosin 3 (Tpm3) that could discriminate CRC at a significant level (p = 0.0146). Our results suggest that at least one of the identified proteins, Tpm3, could be used as a biomarker in the early detection of CRC, and further studies should provide unequivocal evidence for the real-life clinical validity and usefulness of Tpm3.

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Affinity Capture Enrichment versus Affinity Depletion: A Comparison of Strategies for Increasing Coverage of Low-Abundant Human Plasma Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms21165903 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5903

Author(s):

Nicolai Bjødstrup Palstrøm ◽

Lars Melholt Rasmussen ◽

Hans Christian Beck

Keyword(s):

Mass Spectrometry ◽

Small Molecule ◽

Plasma Proteins ◽

Biomarker Discovery ◽

Plasma Samples ◽

Label Free ◽

Affinity Capture ◽

Liquid Chromatography Tandem Mass ◽

Removal System ◽

Equalization Method

In the present study, we evaluated four small molecule affinity-based probes based on agarose-immobilized benzamidine (ABA), O-Phospho-L-Tyrosine (pTYR), 8-Amino-hexyl-cAMP (cAMP), or 8-Amino-hexyl-ATP (ATP) for their ability to remove high-abundant proteins such as serum albumin from plasma samples thereby enabling the detection of medium-to-low abundant proteins in plasma samples by mass spectrometry-based proteomics. We compared their performance with the most commonly used immunodepletion method, the Multi Affinity Removal System Human 14 (MARS14) targeting the top 14 most abundant plasma proteins and also the ProteoMiner protein equalization method by label-free quantitative liquid chromatography tandem mass spectrometry (LC-MSMS) analysis. The affinity-based probes demonstrated a high reproducibility for low-abundant plasma proteins, down to picomol per mL levels, compared to the Multi Affinity Removal System (MARS) 14 and the Proteominer methods, and also demonstrated superior removal of the majority of the high-abundant plasma proteins. The ABA-based affinity probe and the Proteominer protein equalization method performed better compared to all other methods in terms of the number of analyzed proteins. All the tested methods were highly reproducible for both high-abundant plasma proteins and low-abundant proteins as measured by correlation analyses of six replicate experiments. In conclusion, our results demonstrated that small-molecule based affinity-based probes are excellent alternatives to the commonly used immune-depletion methods for proteomic biomarker discovery studies in plasma. Data are available via ProteomeXchange with identifier PXD020727.

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Evaluation of soluble KIT (sKIT) as a potential surrogate marker for TTP in sunitinib malate (SU)-treated patients (pts) with advanced GIST

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.10007 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 10007-10007 ◽

Cited By ~ 2

Author(s):

M. Blackstein ◽

X. Huang ◽

G. D. Demetri ◽

P. G. Casali ◽

C. R. Garrett ◽

...

Keyword(s):

Kinase Inhibitor ◽

Surrogate Marker ◽

Sampling Period ◽

Clinical Results ◽

Phase Iii ◽

Plasma Samples ◽

Double Blind ◽

Cox Models ◽

Time Changes ◽

The Impact

10007 Background: SU is an oral, multitargeted tyrosine kinase inhibitor of KIT, PDGFRs, VEGFRs, RET and FLT3, approved multinationally for the treatment of imatinib (IM)-resistant/-intolerant GIST. Preliminary analysis in a SU phase I/II GIST study suggested that a decline in plasma sKIT levels may correlate with measures of clinical benefit. We evaluated the potential of sKIT as a surrogate marker for TTP using samples obtained in a randomized, double-blind, placebo-controlled phase III study of SU in pts with IM-resistant/-intolerant GIST, clinical results of which have been reported previously. Methods: 312 pts were randomized (2:1) to receive SU 50 mg (n=207) or placebo (n=105) daily in 6-wk cycles (4 wks on treatment, 2 wks off), respectively. The primary endpoint was TTP as per RECIST. Levels of sKIT in plasma samples were measured in cycle 1 on days 1, 14 and 28 and in cycles 2 and 3 on days 1 and 28 using a performance-validated ELISA. Prentice Criterion, Cox models and the Proportion of Treatment Effect (PTE) were used to analyze the results (PTE of 1 = ideal surrogate). Results: Numbers of pts with matched pairs of baseline and on-study plasma samples varied from 228 pts at cycle 1, day 14 to 106 pts at the end of cycle 3. After 4 wks of treatment (cycle 1, day 28), plasma sKIT levels began to exhibit a significant decrease (P<0.0001) in response to SU treatment; at the same time, changes in the level of sKIT (decreases vs. increases) also became indicators of TTP (HR=0.53; 95% CI, 0.37–0.75; P=0.0003), with decreases associated with longer TTP. This trend continued throughout the sampling period, with the effect persisting off treatment. Although the impact of SU on plasma sKIT levels continued to be seen after 2 cycles, sKIT changes became a better indicator of TTP than initial treatment group by cycle 2, day 28 (PTE = 0.62; HR=0.51; 95% CI, 0.38–0.69; P<0.0001), and at cycle 3, day 1 (PTE = 0.64; HR=0.42; 95% CI, 0.29–0.60; P<0.0001). Conclusion: These preliminary findings suggest that circulating sKIT may be a surrogate marker for TTP in GIST pts after 2 cycles of SU treatment. Further studies are warranted to confirm these findings and to establish whether sKIT can be used as a general surrogate marker of clinical outcomes in GIST pts with SU or other therapies. [Table: see text]

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Impact of the combination of durvalumab (MEDI4736) plus olaparib (AZD2281) administered prior to surgery in the molecular profile of resectable urothelial bladder cancer: NEODURVARIB Trial.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.6_suppl.542 ◽

2020 ◽

Vol 38 (6_suppl) ◽

pp. 542-542 ◽

Cited By ~ 5

Author(s):

Juan Francisco Rodriguez-Moreno ◽

Guillermo de Velasco ◽

Inmaculada Bravo Fernandez ◽

Carlos Alvarez-Fernandez ◽

Ricardo Fernandez ◽

...

Keyword(s):

Clinical Trial ◽

Adverse Events ◽

Molecular Characterization ◽

Biomarker Discovery ◽

Checkpoint Inhibitors ◽

Parp Inhibitor ◽

Neoadjuvant Treatment ◽

Molecular Profile ◽

Efficacy And Safety ◽

The Impact

542 Background: Cisplatin-based chemotherapy remains the perioperative treatment in muscle-invasive bladder carcinoma (MIBC). Recent evidence suggests that immune checkpoint inhibitors could be incorporated in this setting. Olaparib is a PARP inhibitor with well-established activity in HRD tumor. Results from trials assessing the combination of durvalumab and olaparib suggest a synergistic effect. However, a molecular characterization is crucial to warrant a rational development. Methods: A phase II clinical trial was designed to assess the impact of neoadjuvant treatment with the combination of durvalumab plus olaparib in the molecular profile of MIBC (NCT03534492; SOGUG-2017-A-IEC(VEJ)-2). Efficacy and safety were secondary objectives. Subjects with cT2-T4a MIBC aimed for cystectomy were treated during 6 to 8 weeks pre-cystectomy. Diagnostic and surgical samples, pre and postreatment blood samples have been collected for the molecular analysis. We present results regarding efficacy and safety. Results: From November 2018 to October 2019 28 patients have been enrolled. 52%/48% of patients had PS 0/1. Median age was 70. TNM stage was: pT2 in 73,6% patients, pT3 in 10.6%, pT4 in 15.8% and 10.6% presented nodal spread. 13 patients have completed neoadjuvant treatment so far and 12 have undergone cystectomy. A wound dehiscence and one death related to surgical procedures were postoperative complications. Pathological complete response rate is 44,5%. Radiological evaluation is ongoing. 10 serious adverse events non-treatment related have been communicated. Any grade of toxicity has been reported in 91% of patients but adverse events grade 3-4 was detected in only 8.3% of cases. Grade 1 pruritus was the unique IR adverse event described in one patient. PARP inhibitors-related adverse events were grade 1 nausea and vomiting (25%), and grade 1 anemia (25%). Conclusions: Preliminary clinical data suggest that Durvalumab in combination with Olaparib could be active and well-tolerated neoadjuvant treatment of MIBC. Molecular characterization and biomarker discovery will be presented separately. Clinical trial information: NCT03534492.

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