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A Novel Strategy for Large-Scale Metabolomics Study by Calibrating Gross and Systematic Errors in Gas Chromatography-Mass Spectrometry Yanni Zhao, Zhiqiang Hao, Chunxia Zhao, Jieyu Zhao, Junjie Zhang, Yanli Li, Lili Li, Xin Huang, Xiaohui Lin, Zhongda Zeng, Xin Lu, and Guowang Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03912 • Publication Date (Web): 12 Jan 2016 Downloaded from http://pubs.acs.org on January 18, 2016

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Analytical Chemistry

A Novel Strategy for Large-Scale Metabolomics Study by Calibrating Gross and Systematic Errors in Gas Chromatography-Mass Spectrometry

Yanni Zhao1, Zhiqiang Hao2, Chunxia Zhao1, Jieyu Zhao1, Junjie Zhang1, Yanli Li1, Lili Li1, Xin Huang2, Xiaohui Lin2, Zhongda Zeng1,Xin Lu1*, Guowang Xu1*

1

Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of

Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. 2

School of Computer Science & Technology, Dalian University of Technology,

Dalian 116023, China.

* Address correspondence to: Prof. Dr. Guowang Xu, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Phone / Fax: +86-411-84379530. E-mail: [email protected]. Prof. Dr. Xin Lu, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Phone / Fax: +86-411-84379559. E-mail: [email protected].

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ABSTRACT:

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Metabolomics is increasingly applied to discover and validate metabolite

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biomarkers and illuminate biological variations. Combination of multiple analytical

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batches in large-scale and long-term metabolomics is commonly utilized to generate

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robust metabolomics data, but the gross and systematic errors are often observed. The

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appropriate calibration methods are required before statistical analyses. Here, we

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develop a novel correction strategy for large-scale and long-term metabolomics study,

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which could integrate metabolomics data from multiple batches and different

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instruments by calibrating gross and systematic errors. Gross error calibration method

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applied various statistical and fitting models of the feature ratios between two

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adjacent quality control (QC) samples to screen and calibrate outlier variables. Virtual

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QC of each sample was produced by a linear fitting model of the feature intensities

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between two neighboring QCs to obtain a correction factor and remove the systematic

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bias. The suggested method was applied to handle metabolic profiling data of 1197

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plant samples in nine batches analyzed by two gas chromatography-mass

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spectrometry instruments. The method was evaluated by the relative standard

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deviations of all the detected peaks, the average Pearson correlation coefficients and

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Euclidean distance of QCs and non-QC replicates. The results showed the established

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approach outperforms the commonly used internal standard correction and total

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intensity signal correction methods, it could be used to integrate the metabolomics

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data from multiple analytical batches and instruments, and allows the frequency of

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QC to one injection of every 20 real samples. The suggested method makes a large

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amount of metabolomics analysis practicable.

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Keywords: metabolomics, large-scale, gross error, systematic bias

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Analytical Chemistry

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 INTRODUCTION

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Metabolomics is an indispensable branch of systems biology research which is

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focused on the holistically quantitative and qualitative investigations of the changes of

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the low-molecular-weight metabolites (≤ 1500 Da) related to genetic, environmental

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and disease perturbations in biological samples.1-3

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Chromatography coupled with mass spectrometry (MS) and nuclear magnetic

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resonance (NMR) spectroscopy are the most commonly applied tools in metabolomics

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study.4-7 Among them, gas chromatography-mass spectrometry (GC-MS) could

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efficiently separate and detect some of hydrophilic endogenous metabolites (e.g.,

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sugar, amino acids and organic acids) after derivatization,8 which are essential

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substances to maintain the life activities.9,10 To identify and validate the physiological

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perturbations and metabolite biomarkers of different living systems in the primary

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metabolome, large amount of samples are to be analyzed using a high-throughput

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approach based on GC-MS platform.11 In our previous work, a pseudotargeted

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metabolomics approach has been developed in which the characteristic ion

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information obtained from the nontargeted metabolomics data without the aid of

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standard references was used to acquire the metabolome data by using the selective

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ion monitoring (SIM) GC-MS. It displayed wider linear range, higher sensitivity and

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better repeatability than nontargeted GC-MS in the full-scan mode.10,12 A stability and

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repeatability platform is required for the analysis of large sample sizes, unfortunately,

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until now, it is still not easy to analyze all samples in an analytical batch due to MS

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signal drift in long-term metabolomics studies. It is often necessary to split large-scale

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samples into multiple batches.13 The occurring trouble is integration of these data

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because the gross error and systematic bias are often met in metabolomics data of

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multiple batches and they were caused by instrument cleaning and calibration, 3

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accessories changing, etc.

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In the previous studies,13 both the internal standard (IS) correction and total

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intensity signal correction (TISC) were considered as sample-based signal calibration

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procedures, which utilized a correction factor (e.g., IS and TISC) of each sample to

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normalize all of the analytes in a given sample.10,14 Ideally, the IS or TISC should

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display similar variation tendency to all of the detected metabolites for the removal of

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systematic drifts. Actually, for cases in which hundreds to thousands of analytes are

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detected, it is impossible that TISC or IS shows similar changes in signal patterns to

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all the metabolic features. To solve this problem, some novel tactics of feature-based

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signal correction have been suggested to calibrate systemic errors in metabolomics

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study.13,15,16 Wang et al. utilized a single value regression model with the total

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abundance information of each sample to develop a Batch Normalizer method, which

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could remove both batch and injection order effects.16 Kamleh et al.13 employed the

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local or global mean intensity of QCs (LoMec and GoMec) and the local or global

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linear regression of QC intensity values (LoReg and GoRec) to formulate a correction

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factor of each feature and further remove the systematic bias. These suggested

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protocols showed a remarkable improvement in repeatability compared to TISC and

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IS methods. Fages et al.15 proposed a grouped-batch profile (GBP) calibration method

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using the systematic bias of feature in each batch to calibrate the actual metabolic

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response. This established procedure could adjust systematic variation of NMR

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metabolomic data sets and improve the prediction of multivariate regression models

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for robust classification of biological samples. However, all of these methods were

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concentrated on the systematic bias calibration, few were on gross errors correction.

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Actually, both of gross and systematic errors are to be calibrated in order to obtain a

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high quality metabolomics data. 4

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Analytical Chemistry

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In this study, a GC-MS-based pseudotargeted metabolomics approach was

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applied to acquire the metabolic profiling data of plant leaves. A novel calibration

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strategy was developed for the integration of large-scale and long-term metabolomics

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data, in which both systematic and gross errors could be corrected. A metabolomics

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data set with 1197 plant leaf samples including 130 QCs in nine batches analyzed by

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using two different instruments were applied to evaluate this suggested calibration

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method by calculating the percentages of features (POF) with relative standard

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deviation (RSD) less than 15%, the average Pearson correlation coefficients (r) and

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the Euclidean distance (ED) of the replicates in principal components analysis (PCA)

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scores plots. Additionally, the frequency of QC injection was also investigated.

86 87

 EXPERIMENTAL SECTION

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Strategy description

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In large-scale GC-MS metabolomics analysis, some necessary instrumental

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maintenances (e.g., changing accessories and columns, cleaning ion source and tuning

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instrument), are frequently performed to minimize the signal intensity drifts caused by

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instrument contamination and column age changes. Additionally, several batches are

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analyzed due to the different sample collection times or repeatability requirement of

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the instrument. Sometimes data obtained from different GC-MS instruments within a

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laboratory or inter-laboratory were to be integrated or compared. In considering above

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these cases, we design an experiment procedure to analyze 1197 plant leaves

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including 130 QCs in nine batches (Figure 1). The samples in the first four batches

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were consecutive injection and some changes in instrument accessories (e.g., the

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injection port liner and silicone septum) between two batches were conducted to avoid

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the signal decrease. After a long time intervals between batch 4 and batch 5, batch 6 5

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and batch 7, and batch 8 and batch 9, the MS detector was tuned and the most suitable

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instrument parameters (e.g., detector voltage, RF Gain, RF offset and lens voltage)

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were renewed and used to analyze samples. The samples in the first seven batches and

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last two batches were acquired by GC-QP2010 Plus and GC-MS QP2010,

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respectively. 188, 209, 25, 25, 156, 97, 354, 84, and 59 samples were analyzed in

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batches 1, 2, 3, 4, 5, 6, 7, 8 and 9, respectively. The same QC samples and five kinds

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of non-QC replicates (i.e., S1, S2, S3, S4 and S5) were analyzed in each batch to

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evaluate the data quality and calibration performance. S1, S2, S3, S4 and S5 consisted

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of 43, 43, 31, 24 and 36 fresh tobacco leaf samples, respectively.

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Systematic and gross errors calibration methods were developed using the data of

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tobacco leaves from first four batches. Systematic error calibration method was

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established by inserting the virtual QCs to the linear fitting model of feature intensity

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of two neighboring QCs to correct the each feature in every sample. The gross error

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correction was conducted by calculating the ratio of each variable between adjacent

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QCs to screen outliers and establishing a linear fitting model to correct the outliers.

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Additionally, the developed method was further applied to correct data of all the 1197

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samples, and the calibration performance was evaluated by investigating the RSD

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values of peak intensity, the average ED and r of the samples (Figure S-1 in the

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Supporting Information).

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Experimental

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Chemicals. Acetonitrile and isopropanol were HPLC-grade from Merck

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(Darmstadt, Germany). Ultrapure water was obtained by filtration of distilled water

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using a Milli-Q system (Boston, USA). Pyridine, methoxyamine hydrochloride and

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MSTFA

(N-methyl-N-(trimethylsilyl)-trifluoroacetamide)

used

as

GC-MS 6

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derivatization reagents were purchased from Sigma-Aldrich (St. Louis, USA). Internal

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standards (ISs), tridecanoic acid and vanilic acid, from Sigma-Aldrich (St. Louis,

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USA) were used to monitor repeatability of sample pretreatment and instrument.

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Damascenone, alpha-ionone, methyl palmitate, methyl linoleate and methyl oleate

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used as external standards (ESs) to monitor stability of the instrument, were obtained

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from Alfa Aesar (Ward Hill, USA).

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Sample Preparation. A total of 1197 fresh tobacco leaf samples including 130

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QC samples were acquired from three main growing districts in China, including

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Henan, Yunnan and Guizhou provinces. All of fresh leaves were ground to

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homogeneous powders and stored at -80 oC until metabolite extraction. The equal

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amount of analytical samples was blended for preparation of the QC to monitor the

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analytical method and correct the large-scale GC−MS metabolomics data.

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The preparation workflow of the leaf samples was similar to our previous work

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with a few modifications.10 About 10 mg leaf powder was soaked in 1.5 mL of the

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isopropanol/acetonitrile/water (3:3:2, v/v/v) mixed solution (including 20 μL of 0.1

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mg/mL internal standards and external standards solutions) and adequately

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vortex-mixed for 4 min to extract leaf metabolites using a Multi-Tube Vortexer

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(VX-2500). After centrifugation at 14 000 rpm for 10 min at 4 °C, 500 μL of

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supernatant was collected in a new Eppendorf tube, and then freeze-dried using a

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Labconco centrifugal concentrator for six hours. The dried residue was immersed in

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100 μL of 20 mg/mL methoxyamine pyridine solution in 37 oC water bath for 90

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min to protect the ketones and aldehydes. Subsequently, a 60 min silylation reaction

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was performed by adding 80 μL of MSTFA into the above solution in 37 oC water

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bath to increase the volatility of the metabolites. Ultimately, the derivatized sample

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was transferred to a glass vial with a conical insert for GC-MS analysis. 7

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GC-MS Pseudotargeted Method. The samples were analyzed by two different

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GC-MS instruments (QP 2010 and QP 2010 Plus, Shimadzu, Japan) using two

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different DB-5 MS fused silica capillary columns (30 m × 0.25 mm × 0.25 µm,

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Agilent, USA) in the constant flow mode with 1.2 mL/min high-purity helium

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(99.9995%) as the carrier gas. The oven temperature was set to 70 oC, maintained for

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3 min and then increased at 5 oC/min to 310 oC, held for 5 min. The split ratio was set

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as 10:1 and the interface temperature was adjusted to 280 oC. The electron impact (EI)

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at 70 eV was used as the ionization mode. The temperature of inlet and ion source

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were 300 oC and 240 oC, respectively. The scope of mass signal acquisition under

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full-scan mode was set to 33-600 m/z with a scan rate of 5 scans/s.

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To establish a pseudotargeted GC-MS method,10,12 the raw data at full-scan mode

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of a double concentration QC sample was deconvoluted for peak identification using

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the AMDIS from the NIST (Gaithersburg, USA) and LECO ChromaTOF (St. Joseph,

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USA) software programs. Subsequently, the selection of the characteristic ions of the

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metabolic features was performed by calculating the ions characteristic values, which

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can reflect the ion uniqueness among the co-eluted components,12 using self-designed

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software and commercial programs (e.g., AMDIS and ChromaTOF). A metabolic

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feature is defined as a detected chromatographic peak and a single metabolite can

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have multiple metabolic features.11 The SIM scan group information was obtained by

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setting the threshold value of the chromatographic retention time (RT) differences

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between two adjacent peaks as 0.1 min. According to the group information, RT and

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characteristic ions of metabolites, the SIM acquired method and quantitative approach

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of the pseudotargeted GC-SIM-MS were established to analyze the tobacco leaves and

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obtain the peak integration table for the further study. The instrument parameters of

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pseudotargeted GC-MS were the same as those of full scan method, except for the MS 8

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acquisition mode. Ultimately, 319 metabolites divided into 50 groups were detected

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by the established pseudotargeted metabolomics method.

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Performance Evaluation

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The performance of different correction methods could be evaluated using PCA,16

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which has frequently been applied to extract the main information from

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high-dimensional data to assess the similarity of analytical samples and the

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repeatability of analytical method. The parameters R2X value of PCA score plot could

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reflect the explained fractions of the total variations. To avoid suffering the

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information loss in the data set, the R2X value of PCA analysis dominated by the

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number of principal components (PCs), was set more than 85%. The corresponding

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PCs were applied to calculate the average ED and r between detected replicates.

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Additionally, the RSD values of the duplicates were another important criterion to

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evaluate the calibration results.13,16 According to the United States Food and Drug

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Administration (FDA) guidance,17 the threshold of RSD for the analytical features

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was set to 15%.

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 RESULTS AND DISCUSSION

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Although pseudotargeted metabolomics method has displayed better repeatability

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compared to nontargeted method,12 its implementation in the integration of large-scale

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metabolomics data still faces significant challenges due to the gross or systematic

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errors derived from MS and/or chromatographic column contaminations and changes

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of instrument accessories and environmental conditions. In order to solve these

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problems, we developed different algorithms to calibrate the gross error and

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systematic bias in different batches and instruments (Figure 1). 9

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Systematic Error Correction

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Systematic bias is one of the primary limited factors for the integration of large

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scale metabolomics data. In this study, we established a Virtual-QC method applying

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a regression model of metabolic abundances of each feature in ω adjacent QC samples

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(ω: the number of QCs) to remove the systematic variance (Figure S-2A in the

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Supporting Information). The intensity value Axij of each metabolic feature j in a

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special sample i was corrected by a correction factor AQCv_ij, which was obtained by

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the linear a fitting of signal intensities of feature j in ω adjacent QC samples. Each

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sample i will have a special AQCv_ij, which was closely related with its inserted position

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(i+1) in a certain fitting line as defined by Eq. 1,

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= a (i +1) + b

AQC

v _i j

(1)

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where a and b are the slope and intercept of the regression line, respectively (Figure

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S-2B in the Supporting Information). The new corrected intensity value Ax'ij was

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calculated according to Eq. 2.

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Ax ′ = ij

Ax

ij

AQC

(2)

v _i j

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Virtual-QC method was compared with the commonly used calibration methods,

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including four kinds of sample-based correction methods (e.g., TISC, TIQSC, IS and

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ES) and five kinds of feature-based signal correction methods (e.g., LoMec, LoReg,

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and GoRec etc.)13 (Table S-1). The average ED, r and RSD values of each peak across

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all QC samples and the non-QC replicates were used to evaluate the calibration

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performances.

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It is important to note that the calibration performances of Virtual-QC method 10

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may be affected by ω values of QCs, which could be optimized according to the data

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set characteristics of the analytical samples. For our metabolomics data, the values ω

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from 2 to 4 displayed the similar calibration performances for QCs and non-QC

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replicates. Hence, the Virtual-QC method at ω = 2 was adopted in the following

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comparison.

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For QC samples, all of systematic error correction (SEC) methods displayed

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distinct improvements in RSD, ED and r compared to the raw data (Figure 2A). The

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RSD values of 48.90% of the metabolites were lower than 15% for the IS1 (tridecylic

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acid) calibration method, which outperformed the other sample-based signal

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correction methods. Nevertheless, three feature-based signal correction procedures

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including LoMec, LoReg and Virtual-QC methods, have the shortest ED, greatest r

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and the strongest repeatability overall with 88.09% POF falling below the 15% RSD

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threshold (Figure 2A). Encouraged by these results, a further optimized work of these

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three feature-based signal correction methods was performed by calculating the

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RSD-value, ED and r of five types of non-QC duplicates (i.e., S1, S2, S3, S4 and S5)

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in first four batches. One interesting observation is that the Virtual-QC approach

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displayed shorter ED, higher r and POF with RSD less than 15% compared to LoReg

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and LoMec methods (Figure 2B). Hence, in the following procedures to study the

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gross error calibration methods, the Virtual-QC approach was employed as a

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systematic bias correction method.

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Gross Error Correction

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Gross error is also an important influencing factor of the correction performance

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and caused by measurement biases, malfunctioning instruments, and so forth.18,19

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Because the intensity value of a metabolic feature Axij was corrected by its 11

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neighboring virtual AQCv_ij based on the Virtual-QC method (Figure S-2 in the

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Supporting Information), the gross error in actual QCs could be transferred to the

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inserted virtual AQCv_ij, further influenced the other test samples and expanded the

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scope of the error distribution. Therefore, the gross error correction needs to be

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conducted before systematic bias calibration. Two vital procedures including

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screening and calibrating outliers, were optimized by comparing the correction

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performances across the various algorithms. In the following optimization procedures,

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the calibration performances of gross error correction method was evaluated by a

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comparison between the gross error plus systematic bias calibration method (G+S)

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and only SEC approach based on Virtual-QC.

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Screening outlier features in QCs. Ideally, the intensity ratio of feature j in two

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adjacent QCs was equal to 1 with consideration of the same QCs analyzed in our

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experiment. Nevertheless, some feature ratios between two neighboring QCs could

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obviously deviate from 1, which probably mainly be induced by the gross errors.

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Hence, we firstly calculated the feature ratios between two adjacent QCs, then four

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different methods (i.e., Each 5%, All-5%, Each-box and All-box) were tested to define

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the potential outlier features (Table S-2 in the Supporting Information). Each-5%

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method calculated 5% of the feature ratios in two adjacent QCs as outliers, other 95%

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metabolites were thought to be highly reliable (Figure S-3A in the Supporting

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Information). All-5% method applied all the ratios to calculate the number of outliers

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based on the 95% metabolites with statistic confidence (Figure S-3B in the Supporting

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Information). During screening process, the ratios of metabolic features were firstly

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sorted from small to large, and then the number of outliers was equally selected from

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two sides of ratio > 1 and < 1. Boxplot is frequently applied to find outliers in data

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through their interquartile range (IQR), the first quartile (Q1) and third quartile (Q3).20 12

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IQR could be calculated by the Q1 subtracted from Q3. Outliers are observations that

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fall below Q1 - 1.5(IQR) or above Q3 + 1.5(IQR). Each group ratio and all ratios

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were used to construct Each-box (Figure S-3C in the Supporting Information) and

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All-box (Figure S-3D in the Supporting Information) models and detect outlier

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features, respectively.

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For QC samples, one interesting observation is that all of the four models of

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screening potential outlier features showed a remarkable improvement in the

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repeatability of QCs with more than 99% POF with RSDs falling within 15% (Figure

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3A). Additionally, the ED-values of QCs in PCA score plots of theses four algorithms

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combined with the SEC method were also shorter than only SEC method. Further

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studies found that Each-5% method displayed the best POF with RSD less than 15%,

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r and the shortest ED than the other methods for five types of non-QC replicates

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(Figure 3B). Hence, we select Each-5% method to screen potential outlier features.

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It should be noted that in Each-5% method 5% of the feature ratios in two

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adjacent QCs were defined as possible outliers, but some of them perhaps have a good

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repeatability and are not necessary to be corrected. Hence, we need further confirm

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whether they are the outlier features which are to be calibrated.

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Firstly, a high quality QC data without outlier variance was needed to further

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confirm outlier features and calibrate the other QCs. In this experiment, a series of QC

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samples in a batch are named as QC0, QC1, …, QCn-1, QCn. Based on the practical

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operation experience, greater gross error possibly is observed in the first QC sample

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of a batch (i.e. QC0) compared with those in other QCs. Consequently, the second QC

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(i.e., QC1) in every batch was firstly corrected to obtain a good quality QC by two

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ratios of AQC /AQC and AQC /AQC (Table S-3 in the Supporting Information). If the ratio

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of feature j in AQC /AQC was outside the threshold value, but was inside the threshold

2

1

3

2j

2

1j

13

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range for the AQC /AQC , we speculated variable j was an outlier for QC1. 3j

2j

301

Two different algorithms (i.e., To-QCn and Former-QCn) were developed to

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further judge the outlier features in the other QCs (Table S-4 in the Supporting

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Information). To-QCn method applied the ratio of feature j in two adjacent QCs to

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confirm the outliers. If the ratio of a feature j in AQC /AQC' was outside the threshold

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value, the variable j was an outlier in QCn. The Former-QCn method applied two

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group ratios of QCs (AQC /AQC and AQC /AQC ) to further select the outlier points.

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If the ratio of a feature j in AQC /AQC was outside the threshold value, but was inside

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the threshold range for the AQC /AQC , we speculated variable j was an outlier for

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QCn. The calibration performances of these two methods on the QCs and other five

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kinds of non-QC replicates were comprehensively compared. For QCs, there were

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100%, 98.75% and 88.09% peaks with RSDs falling within 15% in To-QCn,

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Former-QCn and Virtual-QC, respectively. A shortest ED and highest r in the PCA

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scores were noticed using To-QCn method to adjust the QCs (Figure 3C). Figure 3D

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displayed that To-QCn method also produced the highest r, shortest ED and the

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strongest repeatability profile for the five kinds of non-QC replicates. Therefore,

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To-QCn method was considered as the best performance method for further

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confirming outlier features, which could result in remarkable repeatability

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improvements of the same samples over other methods.

nj

(n+1) j

(n+2) j

nj

(n+1) j

(n+2) j

(n-1)j

(n+1) j

nj

(n+1) j

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Correction of outliers. After the outlier features of QCs were defined, we need

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to establish a model to correct the outlier to a reasonable value. The calibration

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equations for QC0, QC1 and other QCn (n = 2,3,4……) are shown in Eq. 3, Eq. 4 and

322

Eq. 5, respectively.

323

AQC′

0j

 AQC = AQC′ ×  0j 1j  AQC′  1j

   cor r .

(3)

14

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where AQC , AQC' , AQC , and AQC' are the unadjusted QC0, adjusted QC0, unadjusted

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QC1 and adjusted QC1 responses, respectively.

0j

0j

1j

1j

 AQC AQC′ = AQC ×  1j 1j 2j  AQC  2j

326

   cor r .

(4)

327

where AQC , AQC' and AQC are the unadjusted QC1, adjusted QC1 and unadjusted QC2

328

responses, respectively.

1j

1j

AQC′nj = AQC′

329

( n - 1) j

2j

 A QCnj ×  AQC′  (n - 1) j

   cor r .

(n = 2, 3, 4, …)

330

where AQC , AQC' , AQC

331

uncorrected QC(n-1) and corrected QC(n-1) responses, respectively.

n j

(n-1) j

(n-1) j

are the uncorrected QCn, corrected QCn,

To obtain the calibration parameters (i.e., [AQC /AQC' ]corr., [AQC /AQC ]corr. and [AQC

332 333

n j

and AQC'

(5)

0j

j

/AQC'

(n-1) j

1j

1j

2j

n

]corr.) in Eq. 3, Eq. 4 and Eq. 5, we tried eight methods (Table S-5 in the

334

Supporting Information) based on the ratios of the features between two adjacent QCs

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without the outlier features. C1, C2 and C3 methods are based on a linear fitting,

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polynomial fitting of three times and six times, respectively. Additionally, correlation

337

coefficients of polynomial fitting (R2) were used as index to study different methods,

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among them, the thresholds of R2 for C4, C5 and C6 methods were 0.9, 0.95, and 0.99,

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respectively. C7 method directly assigned the lowest and largest threshold values to

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the outliers from the ratios < 1 and > 1, respectively. C8 was established based on

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principle of Virtual-QC. If the peak j of the QCn was outlier, the calibration value AQC'

342

was corrected by the virtual AQC' obtained based on AQC v_nj

(n-1)j

nj

and AQC . (n+1)j

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The performance of the above eight methods for correcting outliers was

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compared by handling metabolomics data of the QCs and non-QC replicates samples

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(S1~S5). Both of C1 and C8 calibrated the QC samples to shorter average ED in the

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PCA score plots compared to other correction methods (Figure 3E). In addition, for 15

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347

most of replicates, the C1 method displayed greater improvement in repeatability

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compared to C8 (Figure 3F). Comprehensively considering the correction

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performance of QCs and the S1~S5 samples, the C1 method was selected as the

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optimal correction outliers method. Detailed gross and systematic errors calibration

351

algorithms were described in the Supporting Information.

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PCA score plots were prepared to visualize the calibration performance of the

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repeatability and similarity of the QC samples. The QC samples of each batch before

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calibration are separated into four parts along the first two PCs of the PCA score plot

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depending on batches despite their intrinsic similarity (Figure 4A). On the contrary,

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the QC samples calibrated by G+S calibration method had a satisfactory performance,

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which were randomly distributed in the PCA score plot, showing the gross error and

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batch systematic biases were evidently reduced (Figure 4B). Furthermore, the

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cumulative RSD curve of QCs peaks indicated that the calibration method based on

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G+S could significantly decrease RSD value of metabolic features in QC samples and

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result in a remarkably improved repeatability compared to the crude data (Figure 4C).

362 363

Frequency of QC Injection

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The frequencies of QC injections could alter the correction factor AQC value,

365

which further influences the performance of each calibration method. In our study, the

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frequencies of QC injections (F) in analytic sequence were set to one in every 5

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analyzed samples (1/5), 1/10, and 1/20. The metabolomics data obtained by IS1, TISC,

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SEC, the optimal G+S method and raw data (RD) without correction, were used to

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define the optimum frequency of QC injections (Figure 4D).

v_ij

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Compared to other methods, G+S method showed the strongest improvement for

371

repeatability with RSDs of all features less than 15% under different F (i.e., 1/5, 1/10, 16

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and 1/20). The second best performing method, SEC method based on Virtual-QC

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algorithm without the calibration of gross error, resulted in 98.75%, 97.81% and

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96.24% of total peak numbers below the 15% RSD threshold at 1/5, 1/10 and 1/20

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frequencies, respectively. Moreover, both of the IS and TISC methods had modest

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calibration abilities, and F didn't affect their calibration results. Nevertheless, the

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lowest frequencies of QC injection (1/20) had a worst performance compared to 1/5

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and 1/10 for uncorrected raw data. F had less effects on the correction results of IS,

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TISC, SEC, and G+S methods. Hence, it is reasonable to keep a F value as 1/20, if

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G+S correction method is used, which had the 100 % POF with RSD less than 15%

381

and could increase the analysis throughput in a given time.

382 383

Method Application in integrating the metabolic profiling data of nine batches of

384

samples

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The number of analytical samples was enlarged to 1197 in nine batches analyzed

386

by two different instruments and columns (Figure 1) to further validate the established

387

G+S method, which was compared with the two commonly used sample-based

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correction methods (i.e. IS and TISC) and two feature-based signal calibration

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methods (i.e. LoReg and LoMec). It was obvious that G+S method calibrated QCs to

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a shortest ED and highest r compared to IS1, TISC, LoReg and LoMec (Figure S-4A

391

in the Supporting Information). Furthermore, the RSDs distribution showed the

392

significant increase in peaks number of QC samples with RSD less than 10%, 15%

393

and 20% compared to IS1, TISC, LoReg and LoMec (Figure S-4B in the Supporting

394

Information). Table 1 shows that G+S method also improved the repeatability of five

395

kinds of non-QC replicates (S1~S5) due to the highest r, the shortest ED of 85% PCs

396

in the PCA scores plot and the largest POF with RSDs within 10%, 15%, 20% and 17

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30%.

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After calibrated by G+S approach, the RSDs of external standard ES1

399

(Damascenon) and internal standard IS1 were decreased from 58.89% to 8.65%

400

(Figure 5A and 5B) and from 62.90% to 12.41% (Figure 5C and 5D), respectively,

401

which also indicates that G+S method can effectively reduce these gross and

402

systematic error influences.

403

The signal response drifts of instrument induced by changes of instrument

404

conditions, can be reflected by the variation tendency of the raw signals of external

405

standard (ES) across different batches, because the same amount of ES was added to

406

sample vial before instrument analysis. The greatest signal changes of instrument

407

were observed between batch 7 and batch 8, which was probably induced by changing

408

GC-MS instrument or column (Figure 5A). The instrument parameter drifts (e.g.,

409

detector voltage, RF Gain, RF offset and lens voltage) by using different tuning files

410

could generate a moderate signal changes by observing the responses drifts of ES1

411

between batches 4 vs 5 and batches 6 vs 7 (Figure 5A). The signal of ES1 from batch

412

1 to batch 4 showed a continuous decline pattern, which was closely related with

413

injection order effects, and the changing glass insert and silicone septum had smaller

414

effects on the signal drifts (Figure 5A).

415

In order to visualize the effects of calibration method on the QCs and the non-QC

416

samples, PCA analysis of 1197 samples based on UV scaling was conducted (Figure

417

5E-F). All of the QC samples and five kinds of the non-QC replicates in nine batches

418

before calibration displayed obvious linear changes in PCA score plot (Figure 6E),

419

while good aggregation results of QCs and non-QC replicates can be obviously

420

observed by the calibration of G+S method (Figure 5F). Moreover, this method can

421

also be used to correct nontargeted metabolomics data. Taking our previous 18

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nontargeted metabolomics data (n=194, one batch) from GC-MS analysis9 as an

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example. After calibrated with the suggested method, QCs (n=16) have shorter ED,

424

higher r and lower RSD value (Figure S-5). The RSD of IS (tridecanoic acid) in 194

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analytical samples was decreased from 8.78% to 7.22%. These data indicate that G+S

426

method can effectively improve the repeatability of nontargeted metabolomics

427

analysis. All the results indicate that the developed G+S method is a universal

428

metabolomics data correction method, which is not only suitable for pesudotargeted

429

but also nontargeted metabolomics data.

430 431



CONCLUSIONS

432

The quality of metabolomics data of large-scale samples set in multiple batches

433

analyzed by different instruments and chromatographic columns could be affected by

434

outliers and systematic errors, which are caused by some unstable environment factors,

435

injection order and batch differences. We developed an novel correction strategy for

436

the integration of large-scale and long-term metabolomics data from pseudotargeted

437

GC−MS by calibrating the outliers and systemic errors and minimizing the frequency

438

of QC injection. Our suggested procedures were as follows,

439

1) correct gross error of large-scale metabolomics data by using Each-5% method

440

to select potential outliers, To-QCn method to further confirm the outlier features, and

441

a linear fitting model of metabolic features to calibrate the outlier variables.

442

2) utilize a feature-based correction algorithm, Virtual-QC, to remove the

443

systematic bias of metabolomics data by using a linear regression model of the

444

intensity values of ω neighborhood QCs of each sample to formulate a correction

445

factor of each feature.

446

3) set the frequency of QC injection in the analytical sequence as one in every 20 19

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447

real samples (1/20) if the metabolomics data are corrected by the above mentioned

448

G+S calibration method.

449 450

It should be emphasized that although the pseudotargeted method was used in this study, the strategy suggested is also suitable to nontargeted analysis.

451 452

 ASSOCIATED CONTENT

453

Supporting Information

454

Additional information as noted in text includes the Algorithm for gross and

455

systematic errors correction, Tables S-1~S5 and Figures S-1~S-5. This material is

456

available free of charge via the Internet at http://pubs.acs.org.

457 458

 AUTHOR INFORMATION

459

Corresponding Authors

460

*E-mail: [email protected] (X.L.); [email protected] (G.W.X.).

461 462

Notes

463

The authors declare no competing financial interest.

464 465

 Acknowledgements

466

This study has been supported by the National Grand Project (2014ZX08012-002) of

467

Science and Technology of China, the project (No. 21375127), key project (No.

468

21435006) and the creative research group project (No. 21321064) from the National

469

Natural Science Foundation of China.

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Table 1. Percentages of features (POF) with RSDs within 10%, 15%, 20% and 30%, Average r and ED using 85% PCs in the PCA analysis of the five types of non-QC replicates (S1, S2, S3, S4 and S5)

Samples Method

S1

S2

S3

S4

S5

Average distance Pearson correlation in PCA coefficient

POF with

G+S

ED 9.53

r 0.84

RSD