Analysis of Bovine Serum Albumin (BSA) Digest on a Thermo Scientific Accucore 150-C18, NanoLC Column

September 1, 2012
Joanna Freeke

,
Thermo Fisher Scientific Inc.

,
Duncan Smith

,
John Griffiths

,
The Paterson Institute of Cancer Research

,
Valeria Barattini

LCGC Asia Pacific

LCGC Asia Pacific, LCGC Asia Pacific-09-01-2012, Volume 15, Issue 3
Page Number: 28

Thermo Scientific Application Note

This application note demonstrates the analysis of trypsin-digested bovine serum albumin (BSA) using a Thermo Scientific Accucore 150-C18 (150 Å pore diameter) nanoLC column.

Accucore™ HPLC columns use Core Enhanced Technology™ to facilitate fast and high efficiency separations. Accucore 150-C18 has been further optimized for the analysis of biomolecules and protein digests by extending the pore size to 150 Å.

The increased pore diameter enables larger peptide fragments to diffuse into the particle and interact with the stationary phase more effectively, resulting in high resolution of these fragments.

Herein, we demonstrate the excellent performance of Accucore 150C18 nanoLC columns for the separation of digested BSA.

Standard Preparation

A 50 fmol/μL solution of digested BSA was prepared.

Instrumentation, Column, Consumables and Method

Thermo Scientific Dionex UltiMate 3000 RSLCnano LC system, coupled to a Thermo Scientific LTQ-Orbitrap XL mass spectrometer fitted with a Proxeon Nanospray Flex ion source. Accucore 150-C18 2.6 μm, 75 μm i.d. × 150 mm nanoLC column (P/N 16126-157569).

Thermo Scientific National Vials and Closures (P/N MSCERT4000-34W).

The sample was loaded directly on the column (1 μL injection volume) through sample loop at gradient start.

Flow rate: 300 nL/min; A: 0.1 % formic acid in water B: 80:20 acetonitrile: water (4–40% B gradient over 30 min; ramp to 95% B over 2 min; hold for 2 min; drop to 4% B over 1 min; hold for 4 min).

Figure 1: Base peak chromatogram of 50 fmol of digested BSA loaded on an Accucore 150-C18 nanoLC column, 75 μm i.d.×50 mm.

Results

Elution of tryptic peptides using the conditions described above was achieved within 36 min (Figure 1). Triplicate analyses showed excellent retention time reproducibility for a set of 12 peptides, with % RSD values below 0.14%. Figure 2 shows the extracted ion chromatograms (EIC) of a subset of the peptides monitored. In all cases the peak shapes were found to be excellent, with minimal peak tailing. A peak capacity value of 200 was obtained (1), showing the high resolution power of Accucore 150-C18 nanoLC columns.

Figure 2: EIC of a set of eight peptides.

Conclusion

Accucore 150-C18 nanoLC columns are an ideal choice for complex proteomic samples, featuring excellent resolution power and run-to-run reproducibility.

References

(1) X. Wang, Anal. Chem. 78(10), 3406–3416 (2006).

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