Optimized Peptide Quantification by LC-MS/MS

Dried peptides were reconstituted in 3% acetonitrile, 97% water, and 0.1% formic acid (volume fraction). Separation and MRM analysis was performed on an Agilent Zorbax Eclipse Plus C18 RRHD column (2.1 mm x 50 mm, 1.8 μm particle) coupled to Agilent 6490 Triple Quadrupole LC/MS system with iFunnel technology (Santa Clara, CA). Peptides were eluted over 30 min gradient from 15% to 35% acetonitrile containing 0.1% (volume fraction) formic acid at a flow rate of 200 μL/min. Acquisition method used following parameters in positive mode: fragmentor 380 V, cell accelerator 4 V, electron multiplier 500 V, and capillary voltage 3500 V. Collision energy was optimized for each peptide using the default equation from Agilent, CE = 0.036 m/z—4.8 [29 (link)]. Dwell times were varied based on complexity of samples being analyzed and were in 80 ms to 120 ms range.

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Anderson K.W., Chen J., Wang M., Mast N., Pikuleva I.A, & Turko I.V. (2015). Quantification of Histone Deacetylase Isoforms in Human Frontal Cortex, Human Retina, and Mouse Brain. PLoS ONE, 10(5), e0126592.

Publication 2015

Zorbax Eclipse Plus C18 RRHD column 6490 Triple Quadrupole LC/MS system

Acetonitrile Capillary Cell Electron Formic acid Peptide

Corresponding Organization : Case Western Reserve University

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Variable analysis

independent variables

Gradient from 15% to 35% acetonitrile containing 0.1% (volume fraction) formic acid
Flow rate of 200 μL/min

dependent variables

Separation and MRM analysis of peptides

control variables

Dried peptides reconstituted in 3% acetonitrile, 97% water, and 0.1% formic acid (volume fraction)
Agilent Zorbax Eclipse Plus C18 RRHD column (2.1 mm x 50 mm, 1.8 μm particle)
Agilent 6490 Triple Quadrupole LC/MS system with iFunnel technology
Acquisition method parameters in positive mode: fragmentor 380 V, cell accelerator 4 V, electron multiplier 500 V, and capillary voltage 3500 V
Collision energy optimized for each peptide using the default equation from Agilent, CE = 0.036 m/z—4.8
Dwell times in 80 ms to 120 ms range

controls

No positive or negative controls were explicitly mentioned in the provided information.

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