The Passport 1200 Sample Prep System enriches peptides and removes matrix interferences for peptide quantitation by LC-MS/MS.

Features

• Peptide extraction and enrichment from 500 Da to 2 kDa
• 96 samples in one hour
• Low-volume loading for precious samples
• Liquid-phase recovery
• Automated operation

Application

Extraction and enrichment of peptides from complex sample matrices such as plasma for quantitation by LC-MS/MS.

Components

The Passport 1200 Sample Prep System consists of the Passport 1200 Sample Prep Station, Cartridges, and Software.

Passport 1200 Station

Passport 1200 Cartridge

Passport 1200 Software Screenshot

How It Works

The Passport Sample Prep Cartridge consists of three layers — the sample well frame, the sample collection tray, and the base. Polyacrylamide gel is precision cast into the bottom of each well in the well frame. A 100-500 MW cut-off dialysis membrane is sealed to the bottom of each collection reservoir in the collection tray. During electrophoresis, samples are separated in the gel based on their electrophoretic mobilities. Those analytes that move faster through the gel are eluted from the tube gel and entrapped into a 50 µL liquid layer in the collection well. Following electrophoretic fractionation, the sample well frame is removed from the cartridge, and each of the fractions in the sample collection tray is easily collected using a standard multiwell pipettor.

Performance

Removal of interferences, reproducible molecular weight cut-offs, and high analyte recoveries enable endogenous peptide quantitation by LC-MS/MS.

Removal of Matrix Interferences:
The Passport 1200 Sample Prep System removes matrix interferences from enriched peptide preparations to improve peptide detection sensitivity and stabilize LC retention times.

Analysis of angiotensin I spiked (100 ng/mL) into human plasma using protein precipitation. Clinical samples are contaminated with many chemical species that interfere with, and can often prevent, the analysis of peptides at endogenous levels. Panel (a) shows a chromatogram of angiotensin I spiked human plasma prepared for analysis by protein precipitation. Co-elution of lipid contaminants with the analyte of interest limits sensitivity of the assay. Attempts to measure endogenous levels of peptides by increasing the starting volume of plasma also increase the level of ion suppression, ultimately decreasing sensitivity (b-d).

Comparison of phospholipid contamination after protein precipitation or preparation using the Passport 1200 System. The chromatogram demonstrates the removal of greater than 99% of the contaminating phospholipids from human plasma samples prepared using Passport 1200 sample preparation. The red trace highlights a representative example, a lyso-phosphatidylcholine (MS/MS transition 496–186). The blue trace is representative of the same plasma sample processed using Passport 1200.

Reproducibility of Electrophoresis:
The Passport 1200 Sample Prep System uses precision-cast polyacrylamide gels, and independent, measured, microprocessor-controlled electrophoretic current delivery to each well, to deliver uniform and reproducible molecular weight cut-offs.

ColorBurst protein standards in the precision-cast
polyacrylamide gels of the Passport 1200 Cartridge

Uniform separation of dye standard across 96 wells
of the Passport 1200 Sample Prep Cartridge.
Samples were run at -30V for 10 minutes.
ColorBurst is a trademark of Sigma-Aldrich Biotechnology LP
and Sigma Aldrich Co., St. Louis, MO.

Peptide Recovery and Quantitation:
Recovery of peptides prepared for quantitation using the Passport 1200 Sample Prep Station is between 85 and 100%, enabling quantitation of endogenous peptides.

Peptide Recovery from Plasma:
Representative chromatograms demonstrating nearly quantitative recovery. Peptides shown above include met enkephalin (red) and leu enkephalin (blue).

Percent Peptide Recovery from Plasma

10 µL plasma spiked with leu enkephalin and met enkephalin (total of 10 ng per peptide) was prepared using the Passport 1200 Sample Prep System and analyzed by LC-MS/MS to determine recoveries in quadruplicate.

Quantitation of endogenous angiotensin I from human plasma

Panel a) shows the measurement of endogenous levels of angiotensin I from 10 µL of human plasma after preparation by Passport 1200 using nanoelectrospray mass spectrometry. The endogenous amount of angiotensin I was determined using standard addition. Panel b) shows the calibration curve used to determine the absolute amount of angiotensin I in the plasma.A concentration of 6.3 ng/mL was measured in this example, which corresponds to 48 amol of angiotensin I in the starting volume of plasma before preparation using the Passport 1200 Sample Prep System. The lower limit of quantitation of the assay is estimated to be less than 1 ng/mL and the lower limit of sensitivity is estimated to be approximately 10pg/mL.

Installation Specifications