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Electrospun nanofibrous samplers (MetaSAMP) provide superior biofluid metabolome stability and coverage following direct laser-assisted rapid evaporative ionization mass spectrometry

Vera Plekhova (UGent) , Margot De Spiegeleer (UGent) , Jozefien Geltmeyer (UGent) , Beata Pomian (UGent) , Varoon Singh (UGent) , Ellen De Paepe (UGent) , Kathleen Wijnant (UGent) , Inge Gies, Nathalie Michels (UGent) , Marilyn De Graeve (UGent) , et al.
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Abstract
INTRODUCTION With the current development of ambient ionization platforms, sample analysis procedures are becoming faster and easier. And yet, sacrificing sample preparation and separation techniques in favor of short analytical run times allows matrix effects to resurface unabated, leading to a decrease in sensitivity accompanied by repeatability and reproducibility issues. In this study, we introduce electrospun polymeric fibrous membranes (MetaSAMPs) for the sampling and direct analysis of biofluids using the established laser-assisted rapid evaporative mass spectrometry (LA-REIMS) methodology (Plekhova et al., Nat.Prot., 2021). By applying customized MetaSAMPs® as a sampling tool and direct substrate for LA-REIMS analysis, we aim to increase the short- and long-term preservation of the metabolome, reduce the impact of macromolecules during analysis while improving metabolome coverage and reproducibility. METHODS Polymeric fibrous sampling membranes (MetaSAMP) were developed by electrospinning with their composition individually optimized for three commonly addressed in light of clinical biochemistry biofluids, i.e. stool, urine and saliva. During validation experiments, pooled biological samples were analyzed as such or impregnated on the MetaSAMP® membranes to compare LA-REIMS signal intensity, metabolome coverage, and reproducibility. Next, the short- and long-term stability were examined by storing the impregnated MetaSAMP® membranes and crude biofluids at 4°C and 20° for 48 hours, or at -20°C and -80°C for up to six weeks. Finally, the clinical applicability of the three biofluid-specific MetaSAMPs® was verified in different pediatric cohorts, i.e. OPERA (n = 95, saliva, 6-16y) and MetaBEAse (n = 76, stool, 6-12y) studying adolescent and childhood obesity and CMA (n = 70, urine, 6 months-3y) studying infant food allergy. PRELIMINARY DATA Based on metabolome coverage, reproducibility and fiber network quality, the optimal urinary MetaSAMP® composition was determined to be a blend of 70/20/10 Polystyrene (PS)/Polyvinylpyrrolidone(PVP)/Polyacrylonitrile(PAN)%, enabling extraction of a broad range of metabolites (log P = -5 to 13). The addition of an outer 10% (w/w) nanofiber PAN coating promoted urine absorption while simultaneously acting as a filter to reduce macromolecule biofouling. Similarly, the optimal compositions of the salivary and rectal MetaSAMPs® were determined as 90/10 PS/PVP and 60/40 PVP/PS, respectively, both with PAN coating. In subsequent validation experiments, impregnated MetaSAMPs showed a higher number of detected metabolic features compared to the crude biofluids whilst maintaining good repeatability with 97%, 66% and 65% of features ≤ 30% CV threshold for the urinary, salivary and rectal MetaSAMP®. After 48h storage, mimicking the maximum transportation time from the patient's home or physician’s office to the laboratory, more metabolic features showed better stability when stored impregnated on the samplers as compared to the raw biofluids, as estimated by the interclass correlation coefficients (ICC). For example, 107 and 732 features detected in urine after storage at 20°C and 4°C, respectively, correlated well between 0 and 48h (ICC > 0.4), while urine impregnated on the MetaSAMP stored at the same conditions generated 1597 and 1851 features with ICCs > 0.4. A similar trend was observed following 6 weeks of storage, as urinary MetaSAMPs still retained 1206 highly correlated features at -20°C, while in urine only 780 remained stable. Lastly, the clinical validation in three independent cohorts showed better predictive power of multivariate OPLS-DA models based on metabolic fingerprints obtained with the MetaSAMPs® , e.g. Q2Y= 0.65 and p-value = 1.3e-11 for urinary MetaSAMP® whereas Q2 Y = 0.52 and p-value = 3.6e-7 for the analysis of crude urinary samples. NOVEL ASPECT Polymeric nanofibrous samplers (MetaSAMPs®) promote biofluid metabolome stability during storage while increasing metabolome coverage and reproducibility following LA-REIMS-based metabotyping.

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MLA
Plekhova, Vera, et al. “Electrospun Nanofibrous Samplers (MetaSAMP) Provide Superior Biofluid Metabolome Stability and Coverage Following Direct Laser-Assisted Rapid Evaporative Ionization Mass Spectrometry.” 70th ASMS Conference on Mass Spectrometry and Allied Topics, Abstracts, 2022.
APA
Plekhova, V., De Spiegeleer, M., Geltmeyer, J., Pomian, B., Singh, V., De Paepe, E., … Vanhaecke, L. (2022). Electrospun nanofibrous samplers (MetaSAMP) provide superior biofluid metabolome stability and coverage following direct laser-assisted rapid evaporative ionization mass spectrometry. 70th ASMS Conference on Mass Spectrometry and Allied Topics, Abstracts. Presented at the The 70th ASMS Conference, Minneapolis, MN, USA.
Chicago author-date
Plekhova, Vera, Margot De Spiegeleer, Jozefien Geltmeyer, Beata Pomian, Varoon Singh, Ellen De Paepe, Kathleen Wijnant, et al. 2022. “Electrospun Nanofibrous Samplers (MetaSAMP) Provide Superior Biofluid Metabolome Stability and Coverage Following Direct Laser-Assisted Rapid Evaporative Ionization Mass Spectrometry.” In 70th ASMS Conference on Mass Spectrometry and Allied Topics, Abstracts. Minneapolis, MN, USA.
Chicago author-date (all authors)
Plekhova, Vera, Margot De Spiegeleer, Jozefien Geltmeyer, Beata Pomian, Varoon Singh, Ellen De Paepe, Kathleen Wijnant, Inge Gies, Nathalie Michels, Marilyn De Graeve, Karen De Clerck, and Lynn Vanhaecke. 2022. “Electrospun Nanofibrous Samplers (MetaSAMP) Provide Superior Biofluid Metabolome Stability and Coverage Following Direct Laser-Assisted Rapid Evaporative Ionization Mass Spectrometry.” In 70th ASMS Conference on Mass Spectrometry and Allied Topics, Abstracts. Minneapolis, MN, USA.
Vancouver
1.
Plekhova V, De Spiegeleer M, Geltmeyer J, Pomian B, Singh V, De Paepe E, et al. Electrospun nanofibrous samplers (MetaSAMP) provide superior biofluid metabolome stability and coverage following direct laser-assisted rapid evaporative ionization mass spectrometry. In: 70th ASMS Conference on Mass Spectrometry and Allied Topics, Abstracts. Minneapolis, MN, USA; 2022.
IEEE
[1]
V. Plekhova et al., “Electrospun nanofibrous samplers (MetaSAMP) provide superior biofluid metabolome stability and coverage following direct laser-assisted rapid evaporative ionization mass spectrometry,” in 70th ASMS Conference on Mass Spectrometry and Allied Topics, Abstracts, Minneapolis, MN, USA, 2022.
@inproceedings{01GSSEWPWEM0F5HNB4VDH4VBZ4,
  abstract     = {{INTRODUCTION 

With the current development of ambient ionization platforms, sample analysis procedures are becoming faster and easier. And yet, sacrificing sample preparation and separation techniques in favor of short analytical run times allows matrix effects to resurface unabated, leading to a decrease in sensitivity accompanied by repeatability and reproducibility issues. In this study, we introduce electrospun polymeric fibrous membranes (MetaSAMPs) for the sampling and direct analysis of biofluids using the established laser-assisted rapid evaporative mass spectrometry (LA-REIMS) methodology (Plekhova et al., Nat.Prot., 2021). By applying customized MetaSAMPs® as a sampling tool and direct substrate for LA-REIMS analysis, we aim to increase the short- and long-term preservation of the metabolome, reduce the impact of macromolecules during analysis while improving metabolome coverage and reproducibility.

METHODS

Polymeric fibrous sampling membranes (MetaSAMP) were developed by electrospinning  with their composition individually optimized for three commonly addressed in light of clinical biochemistry biofluids, i.e. stool, urine and saliva.  During validation experiments, pooled biological samples were analyzed as such or impregnated on the MetaSAMP® membranes to compare LA-REIMS signal intensity, metabolome coverage, and reproducibility. Next, the short- and long-term stability were examined by storing the impregnated MetaSAMP® membranes and crude biofluids at 4°C and 20° for 48 hours, or at -20°C and -80°C for up to six weeks. Finally, the clinical applicability of the three biofluid-specific MetaSAMPs® was verified in different pediatric cohorts, i.e. OPERA (n = 95, saliva, 6-16y) and MetaBEAse (n = 76, stool, 6-12y) studying adolescent and childhood obesity and CMA (n = 70, urine, 6 months-3y) studying infant food allergy.

PRELIMINARY DATA

Based on metabolome coverage, reproducibility and fiber network quality, the optimal urinary MetaSAMP® composition was determined to be a blend of 70/20/10 Polystyrene (PS)/Polyvinylpyrrolidone(PVP)/Polyacrylonitrile(PAN)%,  enabling extraction of a broad range of metabolites (log P = -5 to 13). The addition of an outer 10% (w/w) nanofiber PAN  coating promoted urine absorption while simultaneously acting as a filter to reduce macromolecule biofouling. Similarly, the optimal compositions of the salivary and rectal MetaSAMPs® were determined as 90/10 PS/PVP and 60/40 PVP/PS, respectively, both with PAN coating. 
In subsequent validation experiments, impregnated MetaSAMPs showed a higher number of detected metabolic features compared to the crude biofluids whilst maintaining good repeatability with 97%, 66% and 65% of features ≤ 30% CV threshold for the urinary, salivary and rectal MetaSAMP®. After 48h storage, mimicking the maximum transportation time from the patient's home or physician’s office to the laboratory, more metabolic features showed better stability when stored impregnated on the samplers as compared to the raw biofluids, as estimated by the interclass correlation coefficients (ICC). For example, 107 and 732 features detected in urine after storage at 20°C and 4°C, respectively, correlated well between 0 and 48h (ICC > 0.4), while urine impregnated on the MetaSAMP stored at the same conditions generated 1597 and 1851 features with ICCs > 0.4. A similar trend was observed following 6 weeks of storage, as urinary MetaSAMPs still retained 1206 highly correlated features at -20°C, while in urine only 780 remained stable.
Lastly, the clinical validation in three independent cohorts showed better predictive power of multivariate OPLS-DA models based on metabolic fingerprints obtained with the MetaSAMPs® , e.g. Q2Y= 0.65 and p-value = 1.3e-11 for urinary MetaSAMP® whereas Q2 Y = 0.52 and p-value  = 3.6e-7 for the analysis of crude urinary samples. 

NOVEL ASPECT
Polymeric nanofibrous samplers (MetaSAMPs®) promote biofluid metabolome stability during storage while increasing metabolome coverage and reproducibility following LA-REIMS-based metabotyping.}},
  author       = {{Plekhova, Vera and De Spiegeleer, Margot and Geltmeyer, Jozefien and Pomian, Beata and Singh, Varoon and De Paepe, Ellen and Wijnant, Kathleen and Gies, Inge and Michels, Nathalie and De Graeve, Marilyn and De Clerck, Karen and Vanhaecke, Lynn}},
  booktitle    = {{70th ASMS Conference on Mass Spectrometry and Allied Topics, Abstracts}},
  language     = {{eng}},
  location     = {{Minneapolis, MN, USA}},
  pages        = {{3}},
  title        = {{Electrospun nanofibrous samplers (MetaSAMP) provide superior biofluid metabolome stability and coverage following direct laser-assisted rapid evaporative ionization mass spectrometry}},
  year         = {{2022}},
}