class: center, middle, inverse, title-slide # Data Acquisition using Mass Spec ### Max Qiu, PhD </br> Bioinformatician/Computational Biologist </br> <a href="mailto:maxqiu@unl.edu" class="email">maxqiu@unl.edu</a> ### <a href="https://biotech.unl.edu/bioinformatics">Bioinformatics Research Core Facility, Center for Biotechnology</a> </br> <a href="https://ncibc.unl.edu/">Data Life Science Core, NCIBC</a> ### 02-04-2022 --- background-image: url(data:image/png;base64,#https://media.springernature.com/full/springer-static/image/art%3A10.1186%2Fs13024-018-0304-2/MediaObjects/13024_2018_304_Fig1_HTML.png?as=webp) background-size: 75% # Metabolomics Workflow .footnote[ [Shao, Y., Le, W. Mol Neurodegeneration 14, 3 (2019)](https://doi.org/10.1186/s13024-018-0304-2)</br> Copyright © 2021 BioMed Central Ltd ] ??? Experimental design: control all sources of variances, or make sure equal variances where variances cannot be controlled or removed. Sample size and statistical power: power is the probability of detect an effect when the effect is real; choose a sample size that provide sufficient power to detect a certain effect size. Sample collection, storage and extraction * Type of sample: urine, blood, or tissue/cells * Collection: metabolic quenching * Storage: -80 * Extraction: all sample processed the same way; starts at a chilled condition; standardize workflow Sampling bias Technical replicates vs biological replicates Pseudo-replication Quality assurance and quality control --- # Data Acquisition .pull-left[ ### Instrumentation * Separation: LC-, GC-, CE- * Detection: + -MS (Quadrupole, TOF, Ion-trap/Orbitrap) + -MSn (Triple Qua, Qtrap, Q-TOF, Q-Orbitrap) * Others: DI (direct infusion)-, MALDI-, etc... ] .pull-right[ ### Data preprocessing Converting raw MS data to a data matrix (peak intensity table or concentration table, etc.) containing each peak area in each sample * Peak picking (Deconvolution) * Peak alignment * Chemical identification of metabolites ] <!-- --- --> <!-- # Data Acquisition: Instrumentation --> <!-- ### Mass Spectrometry --> <!-- * Potentially thousands of metabolites (low-levels) --> <!-- * Wide coverage --> <!-- * Excellent sensitivity --> <!-- * High resolution --> <!-- * Separation techniques --> <!-- ### Nuclear Magnetic Resonance (NMR) Spectroscopy --> <!-- * Potentially hundreds of metabolites (most abundant) --> <!-- * High-throughput --> <!-- * Quantitative --> <!-- * Reproducible --> --- # [Comparison of NMR and MS](https://www.ebi.ac.uk/training/online/courses/metabolomics-introduction/designing-a-metabolomics-study/comparison-of-nmr-and-ms/) | |NMR |MS | |:--------------------------------|:-----------------------------------------------------------|:----------------------------------------------------------------------------------------------------| |Sensitivity |Low |High | |Reproducibility |Very high |Average | |Number of detectable metabolites |30-100 |300-1000+ (depending on whether GC-MS or LC-MS is used) | |Targeted analysis |Not optimal for targeted analysis |Better for targeted analysis than NMR | |Sample preparation |Minimal sample preparation required |More complex sample preparation required | |Tissue extraction |Not required - tissues can be analyzed directly |Requires tissue extraction | |Sample analysis time |Fast - the entire sample can be analyzed in one measurement |Longer than NMR - requires different chromatography techniques depending on the metabolites analyzed | |Instrument cost |More expensive and occupies more space than MS |Cheaper and occupies less space than NMR | |Sample cost |Low cost per sample |High cost per sample | --- # Data Acquisition: Instrumentation ### Combination of separation (chromatography) and detection (mass spec) .pull-left[ * Separation: LC-, GC-, CE (**capillary electrophoresis**)- * Detection (**mass analyzer**): + -MS (Quadrupole, TOF, Ion-trap/Orbitrap) + -MSn (Triple Qua, Qtrap, Q-TOF, Q-Orbitrap) * Others: DI (direct infusion)-, MALDI-, etc... ] .pull-right[  .footnote[ [Capillary Electrophoresis](https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_%28Analytical_Chemistry%29/Instrumental_Analysis/Capillary_Electrophoresis) ] ] ??? Capillary electrophoresis: an electrokinetic separation method that **separates ions based on their electrophoretic mobility** with the use an an applied voltage. The electrophoretic mobility is dependent upon **the charge of the molecule, the viscosity of the mobile phase, and the atom's radius**. MALDI: matrix-assisted laser desorption/ionization --- # Data Acquisition: Instrumentation .pull-left[ ### Liquid Chromatography * **Components**: pumps, sampler, column, detector ] .pull-right[  .footnote[ [Gradient HPLC system](https://www.waters.com/waters/en_US/Identifying-and-Quantitating-Compounds/nav.htm?cid=10049064&locale=en_US)</br> Copyright © Waters ] ] --- # Data Acquisition: Instrumentation .pull-left[ ### Liquid Chromatography * Components: pumps, sampler, column, detector * **Operation principle**: separation based on adsorption/desorption rate between analytes and stationary phase + Polarity + Electrical charge + Molecular size <img src="data:image/png;base64,#./img/LC_separation.PNG" width="100%" style="display: block; margin: auto;" /> ] .pull-right[  .footnote[ [Gradient HPLC system](https://www.waters.com/waters/en_US/Identifying-and-Quantitating-Compounds/nav.htm?cid=10049064&locale=en_US)</br> Copyright © Waters ] ] ??? In general, three primary characteristics of chemical compounds can be used to create HPLC separations. They are: • Polarity • Electrical Charge • Molecular Size Molecules are separated based on their physical-chemical properties, specifically the adsorption/desorption rate between analytes and stationary phase. Mobile phase carrying analytes serves as competing agents. Analytes: Molecules that are being separated by chromatography Stationary phase: solid adsorbent material packed in the column Mobile phase: Solvents used to carry and elute the analytes from the stationary phase --- # Data Acquisition: Instrumentation .pull-left[ ### Liquid Chromatography * Components: pumps, sampler, column, detector * Operation principle: separation based on adsorption/desorption rate * **Column (stationary phase)**: polarity opposite to your target analytes * **Mobile phase**: polarity same as your target analytes (competing with analytes)  ] .pull-right[  .footnote[ https://www.youtube.com/MrSimpleScience ] ] ??? What is a chromatogram? --- # Data Acquisition: Instrumentation .pull-left[ ### Liquid Chromatography * Components: pumps, sampler, column, detector * Operation principle: separation based on adsorption/desorption rate * Column (stationary phase): polarity opposite to your target analytes * Mobile phase: polarity same as your target analytes (competing with analytes) * **Separation modes**: normal phase for non-polar, reverse phase for polar, ion exchange for charged metabolites, size-exclusion chromatography. ] .pull-right[  ] .footnote[ [HPLC Separation Modes](https://www.waters.com/waters/en_US/HPLC-Separation-Modes/nav.htm?cid=10049076&locale=en_US) ] ??? HPLC **Separation Modes** In general, three primary characteristics of chemical compounds can be used to create HPLC separations. They are: • Polarity • Electrical Charge • Molecular Size First, two primary separation modes based on polarity: normal phase and reversed-phase chromatography. Second, separation mode based on electrical charge: ion-exchange chromotography (IEC). Third, separation mode based on molecular size: size-exclusion chromatography (SEC). --- # Data Acquisition: Instrumentation .pull-left[ ### Liquid Chromatography * Components: pumps, sampler, column, detector * Operation principle: separation based on adsorption/desorption rate * Column (stationary phase): polarity opposite to your target analytes * Mobile phase: polarity same as your target analytes (competing with analytes) * Separation modes: normal phase for non-polar, reverse phase for polar, ion exchange for charged metabolites * **Elution modes**: isocratic (elutent unchanged, one pump) or gradient (eluent changing throughout the run, two pump) ] .pull-right[  .footnote[ [Isocratic HPLC system](https://www.waters.com/waters/en_US/Identifying-and-Quantitating-Compounds/nav.htm?cid=10049064&locale=en_US)</br> Copyright © Waters ] ] ??? Two basic elution modes are used in HPLC. The first is called isocratic elution. In this mode, the mobile phase, either a pure solvent or a mixture, **remains the same throughout the run**. The second type is called gradient elution, wherein the **mobile phase composition changes during the separation**. This mode is useful for samples that contain compounds that **span a wide range of chromatographic polarity**. As the separation proceeds, **the elution strength of the mobile phase is increased to elute the more strongly retained sample components**. --- # Data Acquisition: Instrumentation .pull-left[ ### Liquid Chromatography * Components: pumps, sampler, column, detector * Operation principle: separation based on adsorption/desorption rate * Column (stationary phase): polarity opposite to your target analytes * Mobile phase: polarity same as your target analytes (competing with analytes) * Separation modes: normal phase for non-polar, reverse phase for polar, ion exchange for charged metabolites * Elution modes: isocratic (elutent unchanged, one pump) or gradient (eluent changing throughout the run, two pump) * **Detector**: spectrophotometric (uv/vis, fluorescence), refractive index (RI), evaporative light-scattering (ELSD), electrochemical, **mass spec** ] .pull-right[ <img src="data:image/png;base64,#./img/uv_vis.PNG" width="90%" style="display: block; margin: auto;" /> ] ??? The working principle of the Spectrophotometer is based on Beer-Lambert's law which states that the amount of light absorbed by a color solution is directly proportional to the concentration of the solution and the length of a light path through the solution. Principle of RI detector: measures the ability of mobile phase to bend light, which changes as the composition of the mobile phase changes, such as when solutes from column. Principle of ELSD: mobile phase is evaporated, light directed at the remaining analytes, scattered light is detected. Principle of electrochemical detector: eluents from the column undergo an electrochemical reaction in an ECD cell with an electrode, electrons are transferred, resulting an electrical current that is recorded. --- # Data Acquisition: Instrumentation ### Mass Spectrometry basics <img src="data:image/png;base64,#./img/acquisition_schema_1.PNG" width="70%" style="display: block; margin: auto;" /> While direct-infusion MS (DIMS) can be acquired, coupled **orthogonal separation** is desirable - GC, LC, CE **Separate charged ions (metabolites) in time or space based on their mass-to-charge ratio** ??? * __Inlet__: introduce samples by infusion/injection * __Ionization Source__: convert sample molecules to ions (accepting or losing protons/electrons) * __Mass Analyzer__: separate ions according to mass & charge (electric or magnetic field) * __Detector__: detects and quantifies ions (image current or ion counting) .footnote[ https://masspec.scripps.edu/landing_page.php?pgcontent=whatIsMassSpec ] --- # Data Acquisition: Instrumentation ### Mass Spectrometry: ionization source <img src="data:image/png;base64,#./img/ionization.png" width="100%" style="display: block; margin: auto;" /> .footnote[ Copyright © 2013 [Diogo Ribeiro Demartini, Tandem Mass Spectrometry - Molecular Characterization](https://www.intechopen.com/chapters/44881) </br> [Mass Spectrometry Ionization Sources](https://www.labcompare.com/18-Mass-Spectrometry-Ionization-Sources/) ] ??? There are many types of ionization sources, chemical ionization, electron ionization, photo-ionization, inductively coupled plasma ionization, MALDI, etc. Most commonly used is electrospray ionization (ESI). The ionization process is based on a **liquid dispersion**, and the process takes place following three main steps: * ESI unit is a vacuum containing a nebulizer (a needle), which applies high voltage to a liquid to produce an aerosol, a fine spray of charged droplets (step 1). * Solvent evaporation, bigger droplets become smaller and smaller (step 2) * Finally, ion ejection from the highly charged droplets (step 3). An important advantage of ESI is that it can be easily coupled with LC systems. Matrix-Assisted Laser Desorption/Ionization (MALDI) In MALDI analyses, the **sample must be mixed with matrix and spotted** in a stainless steel plate prior the analysis in the mass spectrometer. The sample is **co-crystallized** with the matrix, which has an essential function in MALDI. The co-crystallized sample is **ionized by short laser pulses**. Subsequently, the ions are accelerated and the time that they spend to flight in a vacuum tube to reach the detector is measured in a TOF (time-of-flight) analyzer. <!-- --- --> <!-- # Data Acquisition: Instrumentation --> <!-- ### Mass Spectrometry: ionization source --> <!-- ```{r, echo=FALSE, out.width="70%", fig.align = 'center'} --> <!-- knitr::include_graphics("ESI_complexity.PNG") --> <!-- ``` --> <!-- ??? --> <!-- After ESI, many different types of ions can be formed simultaneously for a single metabolites, therefore **the complexity of the data is increased by the detection of multiple peaks for each metabolite.** --> --- # Data Acquisition: Instrumentation ### Mass Spectrometry: mass analyzer .pull-left[ * **Quadrupole** * TOF(time-of-flight) * Ion-trap/Orbitrap ] .pull-right[  ] <img src="data:image/png;base64,#./img/Mass_spectrometer_quadrupole.jpg" width="50%" style="display: block; margin: auto;" /> .footnote[ (RIGHT) Copyright © [SHIMADZU](https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/fundamental/mass_analyzers.html)</br> (LEFT) Copyright © [Wikipedia](https://en.wikipedia.org/wiki/Quadrupole_mass_analyzer) ] ??? Quadrupole: consists of four parallel rods. Apply voltages to two pairs of opposite rods. Ions travel through the space within the four rods. **Due to the amount of voltage applied to the rods, only ions of a certain m/z ratio will reach the detector**, while other ions have unstable trajectories and collide with the rods. This permits selection of an ion with a particular m/z ratio or a range of ratios to pass and be detected. --- # Data Acquisition: Instrumentation ### Mass Spectrometry: mass analyzer .pull-left[ * Quadrupole * **TOF(time-of-flight)** * Ion-trap/Orbitrap ] .pull-right[ <img src="data:image/png;base64,#https://www.shimadzu.com/an/sites/shimadzu.com.an/files/d7/ckeditor/an/lcms/support/fundamental/fundamental_lcms_img032.jpg" width="80%" style="display: block; margin: auto;" /> ] .footnote[ Copyright © [SHIMADZU](https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/fundamental/mass_analyzers.html) ] ??? TOF: After we have charged ions, give all charged ion the **same amount of kinetic energy**. TOF **separate ions based on the time it takes for the ions to travel through a flight tube** with known length and reach the detector. Based on classical physics, **ions with lower m/z will travel the fastest and arrive at the detector first** while ions with larger m/z will travel the slowest and arrive at the detector last. --- # Data Acquisition: Instrumentation ### Mass Spectrometry: mass analyzer .pull-left[ * Quadrupole * TOF(time-of-flight) * **Ion-trap**/Orbitrap <img src="data:image/png;base64,#./img/ion_traps.jpg" width="80%" style="display: block; margin: auto;" /> .footnote[ [Yuan Tian, et al. J. Mass Spectrom., 49: 233-240.](https://doi.org/10.1002/jms.3343)</br> Copyright © 2014 John Wiley & Sons, Inc ] ] .pull-right[ <img src="data:image/png;base64,#https://www.shimadzu.com/an/sites/shimadzu.com.an/files/d7/ckeditor/an/lcms/support/fundamental/fundamental_lcms_img034.jpg" width="90%" style="display: block; margin: auto;" /> .footnote[ Copyright © [SHIMADZU](https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/fundamental/mass_analyzers.html) ] ] ??? Ion trap: There are several variations of IT MS, for example the **2D linear quadrupole** IT MS and the **3D ring** IT MS. In this figure is a 2D linear quadrupole IT, which consists of a donut-shaped ring electrode sandwiched between two end-cap electrodes. To measure a spectrum, end-cap electrodes are first grounded, then a **low high-frequency voltage is applied to the ring electrode**. The ions are introduced into the IT MS in a pulse mode, where they are all temporarily trapped inside the electrode. This state, where the ions with varying mass are experiencing stable oscillations. As the voltage increases, the oscillation of the ion becomes unstable, at which time these ions are discharged via the hole in the end-cap electrode. Quadrupole MS systems separate and detect masses by letting oscillating ions pass through the quadrupole to reach a detector, whereas **ion trap MS systems separate and detect masses by discharging ions with unstable oscillations from the system**. --- # Data Acquisition: Instrumentation ### Mass Spectrometry: mass analyzer .pull-left[ * Quadrupole * TOF(time-of-flight) * Ion-trap/**Orbitrap** <img src="data:image/png;base64,#https://www.creative-proteomics.com/images/Q-Exactive-Hybrid-Quadrupole-Orbitrap-Mass-Spectrometer-4.png" width="70%" style="display: block; margin: auto;" /> ] .pull-right[ <img src="data:image/png;base64,#./img/Orbitrap_mass_analyzer.jpg" width="100%" style="display: block; margin: auto;" /> ] .footnote[ (LEFT) Copyright © [Creative Proteomics](https://www.creative-proteomics.com/support/q-exactive-hybrid-quadrupole-orbitrap-mass-spectrometer.html) </br> (RIGHT) Copyright © [Wikipedia](https://commons.wikimedia.org/wiki/File:Orbitrap_mass_analyzer_-_partial_cross-section.JPG) ] ??? Orbitrap: is an ion trap mass analyzer consisting of an **outer barrel-like electrode** and a **coaxial inner spindle-like electrode** that traps ions in an orbital motion around of spindle. Different ions oscillate at different frequencies, resulting in their separation. As shown in Figure, in the Orbitrap mass analyzer, stable ion trajectories combine **rotation** around an axial central electrode with harmonic **oscillations along it** (z-axis). The frequency of these harmonic oscillations along the z-axis depends only on the ion’s m/z and the instrument. --- # Data Acquisition: Instrumentation ### Mass Spectrometry: mass analyzer <img src="data:image/png;base64,#https://api.intechopen.com/media/chapter/44881/media/image2.png" width="70%" style="display: block; margin: auto;" /> .footnote[ Copyright © 2013 </br> [Diogo Ribeiro Demartini](https://www.intechopen.com/chapters/44881) ] ??? It is important to note that **no single mass analyzer is excellent for all analyses**. Therefore, it is important to understand the different principles, features and characteristics of these mass analyzers and choose the one suitable for your needs. Quadrupole: * compact and simple; relatively cheap; good selectivity; well-suited for coupling with LC * limited mass range; low resolution TOF: * high sensitivity; high resolution; excellent mass range * require fast data aquisision; expensive? Ion trap: * small and relatively cheap; high sensitivity; good resolution * limited mass range; limited resolution; limited ion trap volume --- # Data Acquisition: Instrumentation ### Tandem Mass Spectrometry (MS/MS or MS²) * Triple Quadrupole * Quadrupole-TOF * Quadrupole-ion trap (Qtrap) and Quadruple-orbitrap <img src="data:image/png;base64,#./img/MS_MS.png" width="60%" style="display: block; margin: auto;" /> .footnote[ Copyright © [Wikipedia](https://en.wikipedia.org/wiki/Tandem_mass_spectrometry) ] ??? Tandem mass spectrometry is a technique where two or more mass analyzers are coupled together and increase its ability to analyze samples. * Auto MS/MS mode: When a particular ion or a set of ions meet the set conditions (in ms1), MS1 (usually a quadrupole) is in the **selected ion monitoring (SIM)**, and the collision cell (MS2) can fragment ions. Then the fragment ions can be scanned by MS3 and get a mass spectrum. The auto MS/MS mode is commonly used for **qualitative analysis, unknown compounds identification (based on fragmented spectrum) and structure analysis**. * Targeted MS/MS mode: In this mode, **only the ions targeted by users can get mass spectrum**. Similar to the auto MS/MS mode, MS1 is in SIM, and the collision cell can fragment ions. Then the fragment ions can be scanned by MS3 and get a mass spectrum. This mode is useful for the **quantitative analysis, identification of known compounds, and structural elucidation**. --- # Data Acquisition: Instrumentation ### Tandem Mass Spectrometry (MS/MS or MS²) * **Triple Quadrupole** * Quadrupole-TOF * Quadrupole-ion trap (Qtrap) and Quadruple-orbitrap <img src="data:image/png;base64,#https://www.creative-proteomics.com/images/Triple-Quadrupole-Mass-Spectrometry.jpg" width="60%" style="display: block; margin: auto;" /> .footnote[ Copyright © [Creative Proteomics](https://www.creative-proteomics.com/technology/triple-quadrupole-mass-spectrometry.htm) ] ??? The triple quadrupole mass spectrometer (TQMS, or QqQ), is a tandem mass spectrometer made up of two quadrupole mass analyzers, with a (non-mass-resolving) radio frequency–only quadrupole between them, acting as a collision cell for **collision-induced dissociation (CID)** to **fragment the selected precursors/parent ions, and to generate fragment/daughter ions**. Triple Q can be used to explore the molecular structure by fragmentation patterns of the parent ion. Triple Q has good selectivity and relatively cheap but poor accuracy and resolution. --- # Data Acquisition: Instrumentation .pull-left[ ### Tandem Mass Spectrometry (MS/MS or MS²) * Triple Quadrupole * **Quadrupole-TOF** * Quadrupole-ion trap (Qtrap) and Quadruple-orbitrap ] .pull-right[ <img src="data:image/png;base64,#https://www.creative-proteomics.com/images/Agilent-6540-UHD-Quadrupole-Time-of-Flight-Accurate-Mass-Mass-Spectrometer-2.png" width="100%" style="display: block; margin: auto;" /> ] .footnote[ Copyright © [Creative Proteomics](https://www.creative-proteomics.com/support/agilent-6540-uhd-quadrupole-time-of-flight-accurate-mass-mass-spectrometer.htm) ] ??? The quadrupole mass analyzer can separate ions according to their mass-to-charge ratios (m/z) by using the stability of the trajectories in oscillating electric fields. It can **work as a mass filter** to transmit only ions of a selected m/z value to achieve a stable trajectory. Ions with other m/z values collide with the rods or walls and cannot pass the rods. Selected ions are fragmented in collision cell and product ions travel through TOF for analysis. * Tof mode * Auto ms/ms mode * Targeted ms/ms mode --- # Data Acquisition: Instrumentation ### Tandem Mass Spectrometry (MS/MS or MS²) * Triple Quadrupole * Quadrupole-TOF * **Quadrupole-ion trap (Qtrap) and Quadruple-orbitrap** <img src="data:image/png;base64,#https://www.creative-proteomics.com/images/Q-Exactive-Hybrid-Quadrupole-Orbitrap-Mass-Spectrometer-2.png" width="50%" style="display: block; margin: auto;" /> .footnote[ Copyright © [Creative Proteomics](https://www.creative-proteomics.com/support/q-exactive-hybrid-quadrupole-orbitrap-mass-spectrometer.htm) ] ??? The quadrupole rod assembly works as **ion transmission** device with the possibility to **filter** the transmitted ion according to its mass-to-charge ratios. The ions are transferred into the **C-Trap and then injected into the Orbitrap** mass analyzer to get mass spectra. In addition, ions are passed through the **C-Trap into the HCD cell and conduct MS/MS** experiments in combination with the quadrupole mass filter. --- # Data Acquisition: Preprocessing .pull-left[ ### Chromatography coupled with MS spectrum * Reduce data **complexity** * Reduce **ionization suppression** and increases the number of metabolites detected * Provides additional information for metabolite identification (**RT** and **spectra**) ] .pull-right[  .footnote[ Copyright © [Wikipedia](https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry) ] ] ??? Advantages of coupling chromatography with MS: * Reduce data complexity: staggering the arrival of metabolites. * Reduce Ion suppression and increases the number of metabolites detected. Ion suppression: when a component (less volatile) eluted from the high performance liquid chromatography (HPLC) column influences the ionization of a coeluted analyte. Analytes completing against each other for ion efficiency. * Provides more information for metabolite identification, RT and mass spectra. A LC-MS dataset is **three dimensional**, it includes three components for each metabolite feature (1) the retention time, (2) the mass-to-charge ratio and (3) the chromatographic peak area which is related to the concentration of the metabolite. We apply all of these components to construct a data matrix defining each metabolite and each peak area in each sample. --- # Data Acquisition: Preprocessing ### Chromatography coupled with MS spectrum <img src="data:image/png;base64,#https://www.shimadzu.com/an/sites/shimadzu.com.an/files/d7/ckeditor/an/support/gcms/02_1.gif" width="70%" style="display: block; margin: auto;" /> .footnote[ Copyright © </br> [SHIMADZU](https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/gcms/fundamentals/retention/total_ion.html) ] ??? A LC-MS dataset is **three dimensional**, it includes three components for each metabolite feature (1) the retention time, (2) the mass-to-charge ratio and (3) the chromatographic peak area which is related to the concentration of the metabolite. We apply all of these components to construct a data matrix defining each metabolite and each peak area in each sample. **Extracted Ion Chromatogram**: Observed intensities for a chosen MZ, or MZ range, as a function of RT. **Total Ion chromatogram**: total intensity (sum) for each spectra, across complete or specified MZ, as a function of RT. --- class: inverse, center, middle # Next: Data Preprocessing and Processing Slides created via the R package [**xaringan**](https://github.com/yihui/xaringan). .footnote[ [Waters: Beginners Guild to Liquid Chromatography](https://www.waters.com/waters/en_US/HPLC---High-Performance-Liquid-Chromatography-Explained/nav.htm?cid=10048919&locale=en_US) ]