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Plasma Biomarker Profile Alterations during Variable Blood Storage.

To the Editor:

A major confounding factor in the discovery of disease-specific molecular signatures is the variability in handling clinical blood samples. Consequently, novel protein markers of diseases have failed the "iron test" of validation and implementation into clinical practice because, in addition to patient variability, a range of preanalytical parameters contributes to erroneous results.

The temperature of storing, transporting, and processing of whole blood following collection is one factor that has not been standardized. Often, samples are transferred from the local laboratories to centralized biobanks in a cooled environment or even on dry ice.

Recently, we established a UK biobank to conduct research into the quality of organs donated for transplantation [UK QUOD (Quality in Organ Donation)], collecting donor blood samples from 60 UK transplant centers (www.Quod.org.uk). Taking into consideration the logistical challenges in a clinical setting for sample collection and biobanking, we favored whole blood sample handling and processing at ambient temperature (22 [+ or -] 2[degrees]C) before isolation of plasma by centrifugation and subsequent storage at -80[degrees]C.

We explored how the proteome and degradome (proteolytic processing of the proteome) may change when whole blood remains at ambient temperature for 30 min, 8 h, 24 h, and 48 h before plasma preparation. We defined a baseline plasma protein signature that could be used to filter candidate protein markers in future biomarker discovery studies. To this end, 40 mL of blood from 5 healthy individuals was collected according to our research consent policy. EDTA gel vacutainer plasma gel separator tubes (BD, with Pearlescent White Hemogard Closure) were used without proteinase inhibitors, and blood samples remained at ambient temperature for 30 min, 8 h, 24 h, and 48 h before centrifugation (1500g for 15 min at 22[degrees]C) for plasma isolation. No hemolysis was observed in any of the blood samples before or after blood centrifugation or during the period of 48 h at ambient temperature.

Following depletion of the top 14 plasma proteins using a MARS column (Agilent), plasma proteome signatures of all time points were compared by label-free quantitative LC-MS/MS (LFQ LC-MS/MS) [1] (1) and protein degradation profiles were mapped by the protein topography and migration analysis platform (PROTOMAP) (2, 3) at 30 min and 48 h. LFQ LC-MS/MS analysis of the in-solution tryptic digests was performed by nUHPLCMS/MS (nano ultra-high performance LC-MS/MS) coupled to a Q Exactive tandem mass spectrometer (Thermo Scientific), as previously described (1). Quantification of each protein across all time points was performed by PROGENESIS QIP (QI for Proteomics) software v3.1.4003.30577 (Nonlinear Dynamics) based on the peptide intensity at the MS1 level following chromatogram alignment.

Quantification analysis of all time points indicated that proteins were remarkably stable at ambient temperature up to 48 h with no obvious trend of change with time [at least 2 unique peptides and >95% CI in protein identification, (1% false-discovery rate)]. Changes in protein abundance occurred in <5% of the proteome, and 20 proteins showed significantly increased concentrations over time (ANOVA P [less than or equal to] 0.05, Table 1). Amongst these were thrombospondin-1 and cystatin C, both previously defined biomarkers (4, 5). The majority of proteins that were enriched with time were of platelet or leukocyte origin, suggesting a low level of cell activation possibly due to sheer stress of blood extraction from the vascular system during collection.

The two extreme conditions, 30 min and 48 h, were selected to further investigate the full effect of proteolysis by PROTOMAP analysis. Five individual samples per condition were pooled to one, proteins were separated by 1D SDS-PAGE and divided into 22 gel bands per condition. Proteins captured in gel bands were digested and analyzed by LC-MS/MS. PROTOMAP bioinformatics combined information from the 1D gel migration of protein fragments and LC-MS/MS analysis to map the degradation profile for each identified protein (2, 3). 820 unique proteins were identified and 22 proteins demonstrated a clear proteolysis profile with protein fragments identified in lower molecular weight bands in T = 48 h when compared to the "parent protein" identified in higher molecular weight bands in T = 30 min plasma samples. A high number of the protein fragments detected in T = 48 h plasma samples were derived from complement and coagulation factors, indicating partial proteolytic processing upon blood storage possibly due to plasma circulating proteases (Table 1).

In summary, we have produced a reference list of proteins that are significantly altered when whole blood is stored in ambient temperature and demonstrated remarkable few changes in the plasma proteome. This suggests that blood sample storage at ambient temperature maintains proteome integrity for up to 48 h in a logistically challenging environment where samples are donated and collected at multiple hospital sites at any time throughout the day or night.

Author Contributions: AH authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: M. Kaisar, NHS Blood and Transplant Trust Fund TF031; R.J. Ploeg, COPE FP7; B.M. Kessler, Wellcome Trust Award 097813/Z/11/Z and John Fell FundAward 133/075.

Expert Testimony: None declared.

Patents: None declared.

Acknowledgments: We thank Dr. Roman Fischer for his expert input on data analysis and Dr. Regent Lee and Mrs. Sandrine Rendel for their useful comments in revising this manuscript. The mass spectrometry proteomics data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD004205 and 10.6019/PXD004205.

References

(1.) Fischer R, Kessler BM. Gel-aided sample preparation (GASP)-a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells. Proteomics 2015; 15:1224-9.

(2.) Dix MM, Simon GM, Cravatt BF. Global mapping of the topography and magnitude of proteolytic events in apoptosis. Cell 2008; 134:679-91.

(3.) Niessen S, Hoover H, Gale AJ. Proteomic analysis of the coagulation reaction in plasma and whole blood using PROTOMAP. Proteomics 2011; 11:2377-88.

(4.) Starlinger P, Haegele S, Wanek D,Zikeli S,Schauer D, Alidzanovic L, et al. Plasma thrombospondin 1 as a predictor of postoperative liver dysfunction. Br J Surg 2015; 102:826-36.

(5.) McCaffrey J, Coupes B, Chaloner C, Webb NJA, Barber R, Lennon R. Towards a biomarker panel for the assessment of AKI in children receiving intensive care. Pediatr Nephrol 2015; 2015; 30:1861-71.

Maria Kaisar [2,3,6] *

Leon F.A. van Dullemen [2,5] ([dagger])

Marie-Laetitia Thezenas [6] [dagger])

Philip D. Charles (6)

Rutger J. Ploeg (2,3,4) ([dagger])

Benedikt M. Kessler (6) ([dagger])

[2] Nuffield Department of Surgical Sciences University of Oxford, Oxford, UK

[3] NHS Blood and Transplant Hertfordshire, UK

[4] Oxford Biomedical Research Centre Oxford, UK

[5] Surgical Research Laboratory University of Groningen Groningen, The Netherlands

[6] Target Discovery Institute Nuffield Department of Medicine University of Oxford, Oxford, UK

([dagger]) Leon F.A. van Dullemen and Marie-Laetitia Thezenas contributed equally to the work

([double dagger]) Rutger J. Ploeg and Benedikt M. Kessler contributed equally to the work

* Address correspondence to this author at:

Nuffield Department of Surgical Sciences

University of Oxford

OX3 7LJ, UK

Fax 01875-226100

E-mail Maria.Kaisar@nds.ox.ac.uk

Previously published online at DOI: 10.1373/clinchem.2016.257246

[1] Nonstandard abbreviations: LFQ LC-MS/MS, label-free quantitative LC-MS/MS; PROTOMAP, protein topography and migration analysis platform.
Table 1. Preanalytical effects on plasma proteomeand degradome.

                                                LFQ LC-MS/MS

                                               Fold change (a)

                                                 T = 8 h vs
Protein description                              T = 30 min

Fibrinogen gamma chain                               1.4
Apolipoprotein A-I                                   1.3
Profilin-1                                           1.1
DNA polymerase epsilon catalytic subunit A           1.2
Coiled-coil domain-containing protein 11             1.2
Coagulation factor XIII B chain                      1.2
Heat shock protein 70 kDa                            1.3
Dedicator of cytokinesis protein 7                   1.5
Cystatin-C                                           1.3
Uncharacterized protein C2orf53                      1.8
Complement C1q subcomponent subunit B                1.3
Leukocyte immunoglobulin-like receptor               1.2
MCM domain-containing protein 2                      1.6
Ankyrin repeat domain-containing protein 54          2.2
Thrombospondin-1 (b)                                 1.2
Coagulation factor XI (c)
Complement C1r (c)
Actin (c)
Complement C3 (c)
Complement C4B (c0
Talin-1 (c)
Apolipoprotein B-100 (c)
Complement C5 (c)
Ceruloplasmin (c)
Alpha-1 antichymotrypsin (c)
Inter alpha trypsin inhibitor (c)
Kallistratin (c)
Corticosteroid-binding globulin (c)
Serum paraoxonase (c)
Fibronectin (c)
Plasmonogen like protein A (c)
Complement C2 (c)
Fibrinogen alpha chain (c)
Extracellular matrix protein-1 (c)
Collagen alpha 3 (c)
Protein disulphide isomerase A3 (c)

                                                LFQ LC-MS/MS

                                               Fold change (a)

                                                 T = 24 h vs
Protein description                              T = 30 min

Fibrinogen gamma chain                               1.3
Apolipoprotein A-I                                   1.3
Profilin-1                                           2.0
DNA polymerase epsilon catalytic subunit A           1.2
Coiled-coil domain-containing protein 11             1.3
Coagulation factor XIII B chain                      1.2
Heat shock protein 70 kDa                            1.3
Dedicator of cytokinesis protein 7                   1.5
Cystatin-C                                           1.3
Uncharacterized protein C2orf53                      1.9
Complement C1q subcomponent subunit B                1.3
Leukocyte immunoglobulin-like receptor               1.4
MCM domain-containing protein 2                      1.7
Ankyrin repeat domain-containing protein 54          2.3
Thrombospondin-1 (b)                                 1.3
Coagulation factor XI (c)                           N/Ed
Complement C1r (c)                                   N/E
Actin (c)                                            N/E
Complement C3 (c)                                    N/E
Complement C4B (c0                                   N/E
Talin-1 (c)                                          N/E
Apolipoprotein B-100 (c)                             N/E
Complement C5 (c)                                    N/E
Ceruloplasmin (c)                                    N/E
Alpha-1 antichymotrypsin (c)                         N/E
Inter alpha trypsin inhibitor (c)                    N/E
Kallistratin (c)                                     N/E
Corticosteroid-binding globulin (c)                  N/E
Serum paraoxonase (c)                                N/E
Fibronectin (c)                                      N/E
Plasmonogen like protein A (c)                       N/E
Complement C2 (c)                                    N/E
Fibrinogen alpha chain (c)                           N/E
Extracellular matrix protein-1 (c)                   N/E
Collagen alpha 3 (c)                                 N/E
Protein disulphide isomerase A3 (c)                  N/E

                                                LFQ LC-MS/MS

                                               Fold change (a)

                                                 T = 48 h vs
Protein description                              T = 30 min

Fibrinogen gamma chain                               1.2
Apolipoprotein A-I                                   1.3
Profilin-1                                           7.1
DNA polymerase epsilon catalytic subunit A           1.2
Coiled-coil domain-containing protein 11             1.3
Coagulation factor XIII B chain                      1.2
Heat shock protein 70 kDa                            1.2
Dedicator of cytokinesis protein 7                   1.4
Cystatin-C                                           1.3
Uncharacterized protein C2orf53                      1.7
Complement C1q subcomponent subunit B                1.3
Leukocyte immunoglobulin-like receptor               1.4
MCM domain-containing protein 2                      1.7
Ankyrin repeat domain-containing protein 54          2.2
Thrombospondin-1 (b)                                 1.4
Coagulation factor XI (c)
Complement C1r (c)
Actin (c)
Complement C3 (c)
Complement C4B (c0
Talin-1 (c)
Apolipoprotein B-100 (c)
Complement C5 (c)
Ceruloplasmin (c)
Alpha-1 antichymotrypsin (c)
Inter alpha trypsin inhibitor (c)
Kallistratin (c)
Corticosteroid-binding globulin (c)
Serum paraoxonase (c)
Fibronectin (c)
Plasmonogen like protein A (c)
Complement C2 (c)
Fibrinogen alpha chain (c)
Extracellular matrix protein-1 (c)
Collagen alpha 3 (c)
Protein disulphide isomerase A3 (c)

                                               PROTOMAP analysis

                                               T = 48 h vs
Protein description                            T = 30 min

Fibrinogen gamma chain                         No degradation
Apolipoprotein A-I                             No degradation
Profilin-1                                     No degradation
DNA polymerase epsilon catalytic subunit A     No degradation
Coiled-coil domain-containing protein 11       No degradation
Coagulation factor XIII B chain                No degradation
Heat shock protein 70 kDa                      No degradation
Dedicator of cytokinesis protein 7             No degradation
Cystatin-C                                     No degradation
Uncharacterized protein C2orf53                No degradation
Complement C1q subcomponent subunit B          No degradation
Leukocyte immunoglobulin-like receptor         No degradation
MCM domain-containing protein 2                No degradation
Ankyrin repeat domain-containing protein 54    No degradation
Thrombospondin-1 (b)                           Partially degraded
Coagulation factor XI (c)                      Partially degraded
Complement C1r (c)                             Partially degraded
Actin (c)                                      Partially degraded
Complement C3 (c)                              Partially degraded
Complement C4B (c0                             Partially degraded
Talin-1 (c)                                    Partially degraded
Apolipoprotein B-100 (c)                       Partially degraded
Complement C5 (c)                              Partially degraded
Ceruloplasmin (c)                              Partially degraded
Alpha-1 antichymotrypsin (c)                   Partially degraded
Inter alpha trypsin inhibitor (c)              Partially degraded
Kallistratin (c)                               Partially degraded
Corticosteroid-binding globulin (c)            Partially degraded
Serum paraoxonase (c)                          Partially degraded
Fibronectin (c)                                Partially degraded
Plasmonogen like protein A (c)                 Partially degraded
Complement C2 (c)                              Partially degraded
Fibrinogen alpha chain (c)                     Partially degraded
Extracellular matrix protein-1 (c)             Partially degraded
Collagen alpha 3 (c)                           Partially degraded
Protein disulphide isomerase A3 (c)            Partially degraded

(a) The darker shaded part of the table demonstrates proteins that
were identified and quantified by PROGENESIS QI and were compared
across all time points (ANOVA P [less than or equal to] 0.05). A
selection of the proteins identified is included.

(b) Thrombospondin was markedly increased between T = 30 min and later
time points in addition to the generation of proteolytic fragments
observed in T = 48h samples.

(c) Indicates PROTOMAP results. All MS data have been deposited in
the PRIDE repository (PXD004205 and 10.6019/PXD004205).

(d) N/E indicates that the abundance of these proteins was not
changed significantly as measured by LFQ LC-MS/MS. Fragments from
these proteins were identified in the lower molecular weight range by
PROTOMAP analysis.
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Title Annotation:Letters to the Editor
Author:Kaisar, Maria; van Dullemen, Leon F.A.; Thezenas, Marie-Laetitia; Ploeg, Rutger J.; Kessler, Benedik
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Sep 1, 2016
Words:2171
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