Lipids Analytical Tool (LipidAT): automated analysis of lipidomic mass

spectrometry data

Jun Ma

Advisor: Dr. Haixu TangCo-Advisor: Dr. David Wild

Co-Advisor : Dr. Predrag Radivoja

School of Informatics, Indiana University, Bloomington, Indiana

Outline• Introduction to lipidomics and mass spectrometry• Objectives• Data and methods• Results• Future work• Acknowledgements

Lipidomics

• Definition: Large-scale study of pathways and networks ofcellular lipids in biological systems

• Methodology:Identification and quantitation of the thousands ofcellular lipid molecular species and theirinteractions with other lipids, proteins, and othermetabolites

Introduction to Lipidomics

Genome DNA

Transcriptome

Proteome

Metabotome

RNA

Proteins

Sugars NucleotidesAminoacids

Lipids(Lipidome)

Metabolites

Phenotype/FunctionRef: Wikipedia

Phospholipids biological functions

• Key participants in the regulation and control of cellular function

• Bioenergetics, signal transduction• Cell recognition

Structures of phospholipid and membrane bilayer

Phospholipids are amphiphilic with hydrophilic head group and hydrophobic fatty acid chain.Phospholipids are building blocks of cellular membranes.

Ref: Children's Hospital Oakland Research Institute

Alteration of Phospholipids structures

Ref: Phil. Trans. R. Soc. A (2006) 364, 2597-2614

Significance of lipidomics• Change in phospholipid, besides affecting the

absolute amounts of phospholipids per cell, also affect the relative ratios of the various phospholipids species in the membrane, which in turn should lead to changes in the membrane structure and consequently the function.

• Altered phospholipid metabolism has been reported in a variety of diseases, such as anemias, malaria, cancer, nuscular dystrophy, ischemia, diabetes, lung diseases, and liver diseases.

Tandem mass spectrometry approach for phospholipids analysis

• Advantage– Detection, separation and identification of various

phospholipids– Quantization analysis of complex individual

phospholipids in complex mixtures

Phospholipid MS/MS

Objectives of LipidAT

• Identifying and quantifying individual phospholipid species in mixture

• Obtaining a comprehensive picture of the differences in membrane phospholipids between contrasting biological conditions

Workflow of automated processing of MS/MS data by LipidAT

MS2 data

Identification- Generation of peak list- Peak identification by matching m/z of candidate species

Report - Identified lipids species - m/z of precursor ion - m/z and intensity of peak

Quantification -Normalization to define multiple internal standards -Combination of multiple runs

Global Visualization

Heatmap

Species comparison

show

Local Visualization

Customer-Defined

Visualization

Show- std of user-defined peak in multiple- difference in the control and sample

Data Preprocessing

Phospholipids Database -Lipid species library -Fragmentation Information

Loading the raw data• Read raw data (.raw or .mzData file)

– Scan number (integer)– Precursor ion(m/z)– Retention time(min)– Product ions

• m/z (mass-to-charge ratio)• intensity (abundance)

Data preprocessing• Baseline subtraction

– m/z < 100–

• Normalization– Similarity of structures and species– Reference peak picking– Absolute quantification

peak ofintensity :Ipeak of m/z :M 1.0IM

)(

)()(

PXI

XAXI

Ref: Journal of Lipid Research Vol. 42, 2001

A(X): original intensity of peak xI(PX): intensity of reference peak px

Data integration • Reduction the noise and error• MS Data from a series of replicate runs• Weighted moving averaging filter– Reducing random noise at high masses – Retaining a sharp step response– Fitting for time domain encoded signals– Equation:

n

i i

n

i ii

dW

MWA

1

1

Wi: the intensity of peak iMi : the m/z of peak i

Phospholipids Identification • Build-in fragmentation ion database– 9 phospholipids species (GPA,GPCho,GPIns,GPEtn,GPGro,GPSer,Sphingomyelin,Cardiolipin, Lysophospholipids)– 10-30 carbon atoms– 0-6 double bonds– Neutral loss– Allowing negative and positive mode of MS

• Identification standards–Peak must be above threshold–Corresponding peak must have high intensity also in nearby spectra

Visualization

• Heatmap– x-axis: m/z of precursor ion or retention time– y-axis: m/z of fragments– z-axis: color scale coded ratio value of peak

intensity in two contrasting condition • Comprehensive picture

– Difference of components– Difference of absolute quantity of species– Difference of fragments

Heatmap

Heatmap click-on

Fragment ion lookup

Error bar• Visualize the intensity distribution of specific

ion in the sample and control User-defined ions Build-in ions for different PLs species

• Visualize the intensity deviation of the specific ion across several runs – Decide if the combination of multiple runs are

feasible

Maintenance of in–house database

• Database operations– Search data– Insert data– Delete data

• Database integration– Allow biological experts to integrate their prior

data with LipidAT database

Applications & functionalities• Load and view .mzData or .raw data format• Perform batch processing • Display separations, survey scans, and MS/MS data in a

single interface• Access sample reproducibility , evaluate sample quality

and instrument performance• Identify the individual phospholipids in large and complex

datasets • View change of whole phospholipids mixture and

specific peaks in contracting biological conditions• Customize layout to meet the users needs

Future work

• Incorporate other lipids species into database• Identify minor components of lipids mixtures

Acknowledgements• Dr. Haixu Tang• Dr. David Wild• Dr. Predrag Radivojac• Lab mates –

– Quanhu Sheng– Yong Li– Chuanyih Yu

• Linda Hostetter• Cheminformatics and Bioinformatics faculty• School of Informatics• Eli Lilly and Company (funder)