5. AOACSPDSMethods-2018AwardsV3

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Table 2017.12I. Valco valve schedule Time, min Position

Table 2017.12L. Transition parameters Protein analyte Peptide Q1 a

Destination

Q3 b

CE c

0 1 9

A B A

Waste

Cas-1 Cas-2 Cas-3

FFVAPFPEVFGK

692.9 920.5 29 634.4 991.6 33

MS analysis

YLGYLEQLLR

Waste

( j ) Following alkylation, add 4 mL of digestion buffer to each sample. ( k ) Mix by carefully inverting tube multiple times. ( l ) Add 250 µL of the thawed trypsin working solution. ( m ) Mix by carefully inverting tube multiple times. ( n ) Incubate samples overnight in an incubator at 37°C. (Record times at start and finish of incubation.) ( o ) Add 400 µL formic acid to quench the samples. ( p ) Mix carefully as addition of formic acid can cause foaming leading to sample loss. Mix by inverting the tube multiple times. ( q ) Centrifuge samples for 10 min at 4000 g to pellet any precipitate. ( r ) Filter a portion of resulting supernatant using a disposable syringe and 0.22/0.45 µm nylon syringe filter into a 1.5 mL centrifuge tube. ( s ) Thaw an aliquot of the 2000 ng/mL IS spike solution. ( t ) Prepare final sample at 1:10 dilution with 0.1% formic acid and 200 ng/mL IS. ( Example : Add 100 µL of sample, 100 µL of IS spike solution, and 800 µL of 0.1% formic acid to an autosampler vial.) ( u ) Inject 5 µL of sample for analysis. F. Determination ( a )  Column parameters .— See Table 2017.12G . ( b )  Pump gradient .— See Table 2017.12H . Valco valve 2 position schedule .—Position A: Flow from column diverted to waste; position B: flow from column sent to MS for analysis. See Table 2017.12I for schedule. ( c ) Mass spectrometer conditions.— AB Sciex 4000 Q TRAP. See Tables 2017.12J for instrument settings, 2017.12K for analytes, and 2017.12L for transition parameters. ( d ) Sequence format .—( 1 ) DMSO blank, prep blank, solution blank. ( 2 ) MRL samples. HQGLPQEVLNENLLR 880.5 1324.7 51 Lac-1 VYVEELKPTPEGDLEILLQK 1157.1 1453 60 Lac-2 VLVLDTDYK 533.3 853.5 23 Lac-3 LIVTQTMK 467.3 707.4 21 Glu-1 EGSLLLPHYNSR 693.4 773.6 40 Glu-2 GDFELVGQR 510.8 572.5 26 Vic-1 ALPNDVLANAYR 658.9 566.8 26 Vic-2 LQAFEPIR 487.3 732.5 23 Vic-3 GDEFGAFTPIQYK 736.9 1024.8 33 Gly-1 VLIVPQNFVVAAR 713.4 1001.6 33 Gly-2 VFDGELQEGR 575.3 903.4 29 Gly-3 LNALKPDNR 520.8 629.3 32 a  Selected mass in quadrupole 1 (Q1). b  Selected mass in quadrupole 3 (Q3). c  Collision energy (CE).

Table 2017.12J. Instrument settings Fixed Ion transfer voltage IS

5500

Probe temperature Interface heater

TEM

500

ihe

On

Typical

Curtain gas

CUR GS1 GS2 CAD

20 40 50

Ion source gas 1 Ion source gas 2

Collision gas

High

( m )  Needle rinse (95% acetonitrile 5% water) .—Pipet 25 mL ultrapure water into a 500 mL graduated cylinder. Fill to volume with acetonitrile, transfer to a glass bottle, mix well, and degas with sonicator prior to use. E. Preparation of Test Sample ( a ) Thoroughly mix sample prior to weighing. Transfer 500 mg to a 15 mL centrifuge tube. ( b ) Prepare each sample separately to avoid clumping of material in tube. ( c ) Add 10 mL of extraction buffer followed by vortexing for 10–60 s until the sample material is a homogenous slurry. ( d ) Sonicate for 60 min. (While sonicating, preheat the water bath.) ( e ) Spin the tubes down for 10 min at 4000 g to pellet remaining solids. ( f ) Transfer 4 mL of the supernatant by pipet to a new 15 mL conical centrifuge tube and add 150 µL of the thawed reducing solution. ( g ) Vortex samples to mix. Heat for 30 min in a 55°C water bath. ( h ) Cool to room temperature. (While cooling, prepare fresh alkylating solution.) ( i ) Alkylate each sample separately. Add 1.2 mL of the freshly prepared alkylating solution, mix by vortexing, and store the tube protected from light for 30 min at room temperature.

Table 2017.12K. Analytes

Common name

Protein analytes

Species

Protein

Cas-1, Cas-2, Cas-3 Lac-1, Lac-2, Lac-3

Cow (milk) α-S1-casein Cow (milk) β-Lactoglobulin

Bos taurus Bos taurus Oryza sativa Pisum sativum Glycine max

Glu-1, Glu-2

Rice Pea Soy

Glutelin

Vic-1, Vic-2, Vic-3 Gly-1, Gly-2, Gly-3

Vicilin

Glycinin G1

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