california wine lc/ms analysis with sieve 2

California Wine LC/MS Analysis

with SIEVE 2.0

Michael Athanas, Ph.D.

VAST SCIENTIFIC / BRIMS

Mark Dreyer, Ph.D.

ThermoFisher Scientific

B R I M SBiomarker Research Initiative in Mass Spectrometry

Mary LopezDirector

David Sarracino

Manager, Biomarker Workflows

Bryan KrastinsLeader

Biomarker Translational Center

Amol Prakash

Assoc. Director

Informatics Center of Excellence

Michael Athanas

Assoc. Director

Informatics Center of Excellence

Jennifer SuttonInformatics Center of Excellence

Project Manager

brims.center

LC/MS AnalysesMethod Challenge

Screening Requires reproducible

identification by MS or

MS/MS

Discovery Requires existing MS

and MSN databases

Exploration Requires component

detection

Absolute Quantitation Requires calibration

methods

2008 Wildfires and Wine

• Over 2790 individual wild fires

• Weather conditions:

– 3 years of below normal rainfall

– Lightning

• Poor air quality

13 Data Samples

# Blend Location

1 zinfandel Lake

10 petite sirah Lake

13 zinfandel Lake

36 cabernet sauvignon Mendocino

37 petite sirah Mendocino

2 cabernet franc Napa


20 petite verdot Napa

21 cabernet franc Sonoma

25 cabernet sauvignon Sonoma

33 merlot Sonoma

35 merlot Sonoma


http://g.co/maps/nwjfSmall and diverse samples

Sample Processing

13 samples

direct LC

injection

LC/MS using Thermo Q-Exactive

Open Accela 1250

Triplicate measurements interspersed by single matrix blank measurements

Data analysis with SIEVE 2.0

Statistically rigorous automated label-free LC/MS differential analysis platform

Applied to: peptide, protein, small molecule data

State 1

Raw file

State 2

raw file

State …

raw file

Workflow

Align

Detect

Identify

Reports:

•Components

•Identification

•Relative Quantitation

•Statistical Analysis

•Trend information

SIEVE Analysis Platform

SIEVE WORKFLOW

Align Detect Identify

1. Full scan spectra

are typically

acquired at 1Hz to

100Hz with high

mass accuracy

(<5ppm).

Data File 1

Reference

Data File 2

SIEVE Workflow – Alignment 1

Intensity

Intensity

M/Z

M/Z


Intensity

Intensity

M/Z

M/Z


are typically

acquired at 1Hz to

20Hz with high

mass accuracy

(<5ppm).

2. The spectra are

binned.

Data File 1

Reference

Data File 2

X



are typically

acquired at 1Hz to

20Hz with high

mass accuracy

(<5ppm).

2. The spectra are

binned.

3. A dot product

correlation is

calculated between

each pair of spectra

Data File 1

Reference

Data File 2

Scan

# d

ata

file

2

Scan # data file 1


Scan-to-scan

correlation:

Red High

Green Low

•An overlapping tile is constructed

from the next region starting from

the middle of the optimal path.


•An overlapping tile is constructed

from the next region starting from

the middle of the optimal path.

•The full plane is tiled and a final

alignment score is calculated.

Overlapping

measurements

are averaged


Unaligned

basepeaks


Wine

Aligned

basepeaks

Alignment

scores


Wine

Sample - Solvent blank = Analyte signals

Background Subtraction

Adducts, fragments and multimers

[M+H]+ [M+Na]+ [M+K]+

524.3703, z=1, I=4.2E+08, 100% 546.3517, z=1, I=1.0E+08, 24.6% 562.3232, z=1, I=1.1E+06, 0.3%

A+1

Isotopic peaks

525.3730, I=1.2E+08, 28.9%

527.3784, I=3.0E+06, 0.7%

528.3811, I=3.9E+05, 0.1%

A+2 526.3756, I=2.3E+07, 5.5%

A+3

A+4

547.3535, I=2.9E+07, 27.8%

548.3577, I=5.6E+06, 5.4%

549.3595, I=9.0E+05, 0.9%

A+1

A+2

A+3

Isotopic peaks

21.9816

37.9554

Component Detection

Constituents are represented by base component

Frame / Feature

Frame: a well defined rectangular region in the M/Z versus Retention Time plane.

L-Epicatechin

MW = 290.0790

L-Epicatechin

L-Epicatechin

MW = 290.0790

# Blend Location

1 zinfandel Lake

10 petite sirah Lake

13 zinfandel Lake

36 cabernet sauvignon Mendocino

37 petite sirah Mendocino



20 petite verdot Napa

21 cabernet franc Sonoma


33 merlot Sonoma

35 merlot Sonoma


Accurate Mass Identification

Mass accuracy

2.9 ppm

using 445.12

background ion

www.vastsci.com/rawmeat

Local

database

chemspider

web service

Component MW

List of

candidates

Identification Results

• 1224

components

• 255 identified components

• Flavonoid

accurate mass database

MolWt Expression Name

290.079 L-Epicatechin

306.074 Epigallocatechin

314.01 D-glycoside of vanillin

380.1254 Vellokaempferol 3-5-dimethyl ether

382.1047 Velloquercetin 4 -methyl ether

426.0945 Epigallocatechin 3-O-(4-hydroxybenzoate)

436.1153 Epigallocatechin 3-O-cinnamate

450.0793 Quercetin 4 -galactoside

468.1051 Epigallocatechin 3-O-caffeate

472.1 Epigallocatechin 3-O-(3-O-methylgallate)

477.1266 Isorhamnetin 7-alpha-D-Glucosamine;Quercetin 3 -methyl ether 7-alpha-D-Glucosamine;7-[(2-Amino-2-deoxy-alpha-D-glucopyranosyl)oxy]-3-5-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-1-benzopyran-4-one

478.0742 Quercetin 7-glucuronide

486.1157 Epigallocatechin 3-O-(3-5-di-O-methylgallate)

494.0691 Myricetin 3-glucuronide

504.1626 6-Hydroxykaempferol 3-5-7-4 -tetramethyl ether 6-rhamnoside;6-[(6-Deoxy-alpha-L-mannopyranosyl)oxy]-3-5-7-trimethoxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one

516.1262 Kaempferol 3-(3 -4 -diacetylrhamnoside)

552.1474 6-Hydroxymyricetin 3-6-3 -5 -tetramethyl ether 7-glucoside

562.2045 Caohuoside D;8-(3 -Hydroxy-3 -methylbutyl)kaempferol 4 -methyl ether 7-glucoside;7-(beta-D-Glucopyranosyloxy)-3-5-dihydroxy-8-(3-methoxy-3-methylbutyl)-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one

580.1423 Quercetin 3-xylosyl-(1->2)-rhamnoside

600.111 Quercetin 3-(2 -galloylrhamnoside)

610.1317 Quercetin 3-(3 -p-coumarylglucoside)

610.1528 Rutin;3-3 -4 -5-7-Pentahydroxyflavone 3-rutinoside;3-Rutinosylquercetin;Birutan;Quercetin 3-beta-rutinoside;Quercetin 3-O-alpha-L-rhamnopyranosyl-(1->6)-beta-D-glucopyranoside

636.1474 Kaempferol 3-(4 -acetyl-6 -p-coumarylglucoside)

640.127 Quercetin 3-glucuronide-7-glucoside

640.1634 Tamarixetin 3-glucosyl-(1->2)-galactoside;Quercetin 4 -methyl ether 3-glucosyl-(1->2)-galactoside;Thesioideoside;3-[(2-O-beta-D-Glucopyranosyl-beta-D-galactopyranosyl)oxy]-5-7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one

662.163 Kaempferol 3-(2 -3 -diacetyl-4 -p-coumarylrhamnoside

668.1583 Euphorbianin;Quercetin 3-(6 -acetylglucosyl)-(1->3)-galactoside;3-[[3-O-(6-O-Acetyl-beta-D-glucopyranosyl)-beta-D-galactopyranosyl]oxy]-2-(3-4-dihydroxyphenyl)-5-7-dihydroxy-4H-1-benzopyran-4-one

724.1787 Platanoside;Kaempferol 3-(2 -3 -di-(E)-p-coumaroylrhamnoside);3-[[6-Deoxy-2-3-bis-O-[(2E)-3-(4-hydroxyphenyl)-1-oxo-2-propenyl]-alpha-L-mannopyranosyl]oxy]-5-7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one

756.1685 Quercetin 3-(3 -6 -di-p-coumarylglucoside)

770.1841 Kaempferol 3-(3 -p-coumaryl-6 -ferulylglucoside)

772.1845 Quercetin 3-O-beta-(6 -O-E-p-coumaroylglucoside)-7-O-beta-glucoside

….

http://metabolomics.jp

Cluster Wine Flavonoids

Catechin 5-O-(2-feruloyl-6-p-coumaroyl-beta-D-glucopyranoside)

Cyanin

Epigallocatechin

flavonol

Flavylium

L-Epicatechin

Procyanidin

Clustering based upon signal intensity profile

Flavonoid database

Absolute Quantitation

Calibration Curve Method

Dilution series at four concentrations(.02, .04, .2,.4) mg/ml in 10%

methanol

LC/MS using Thermo Q-Exactive

Data analysis with SIEVE 2.0

Epicatechin

Catechin

Orientin

Luteolin

Ellagic Acid

Quercetagetin

Chrysin

Genistein

Rhamnetin

Tamarixetin

Standards

Linear Regression

Observed value in

Zinfandel from Lake

(16ug/ml)

Linear Regression

Observed value in

Cabernet Sauvignon from

Mendocino

(74ug/ml)

Absolute Quantitation

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

mg

/ml

Quercetagetin

L-Epicatechin

Contaminant Response

MolWt FORMULA NAME

209.9406 C7H5Cl3O 2,4,6-Trichloroanisole

150.0681 C9H10O2 2-Methoxy-4-vinylphenol (4-Vinylguaiacol)

168.1514 C11H20O 2-Methylisoborneol

122.0732 C8H10O 4- ethylphenol

152.0837 C9H12O2 4-ethylguaiacol

138.0681 C8H10O2 4-methylguaiacol

108.0575 C7H8O Cresol

164.0837 C10H12O2 Eugenol

96.02113 C5H4O2 Furfural

182.1671 C12H22O Geosmin

124.0524 C7H8O2 Guaiacol

286.1053 C13H18O7 ß-D-glycoside of Guaiacol

314.01 C14H18O8 ß-D-glycoside of vanillin

154.063 C8H10O3 Syringol

Possible smoke related contaminants

Contaminant Response

0.0000

0.0050

0.0100

0.0150

0.0200

0.0250

0.0300

0.0350

0.0400

0.0450

0.0500

2-Methoxy-4-vinylphenol (4-

Vinylguaiacol)

4- ethylphenol

Eugenol

Furfural

Syringol

D-glycoside of Guaiacol

D-glycoside of vanillin

Summary

• More work to be done, more representative samples

• Measuring contaminant free form may likely be insufficient (reference)

Deep appreciation for expert laboratory craftsmanship to:

Mark Dreyer Ph.D., Applications Specialist, ThermoFisher

Thomas Collins, Ph.D, Senior Manager Research and Development, Treasury Wine Estates

For more information:

[email protected]

http://vastscientific.com/sieve

http://vastscientific.com/rawmeat

mailto:[email protected]

http://vastscientific.com/sieve

http://vastscientific.com/rawmeat

california wine lc/ms analysis with sieve 2

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