Page 14 - Application Handbook - Liquid Chromatography

C190-E176

4. Conclusions References
[1] Gorg, A., Weiss, W., Dunn, M. J., Proteomics 2004, 4,
A fully automated 2D RP-LC×RP-LC system, coupled to PDA and 3665–3685.
LCMS-IT-TOF detection was successfully employed for the analysis [2] Rabilloud, T., Proteomics 2002, 2, 3–10. On-line Comprehensive RP-LC×RP-LC/IT-TOF
of α-casein and dephosphorylated α-casein tryptic digests. [3] Beranova-Giorgianni, S., Trends Anal. Chem. 2003, 22, Technical for the Analysis of Proteome Isoforms
Due to the ionic nature of peptides, the use of different pH values 273–281. Report
ensured enough separation selectivity between the two dimensions, [4] Reinders, J., Zahedi, R. P., Pfanner, N., Meisinger, C., A meaningful evaluation tool for native and recombinant proteins
Sickmann, A., J. Proteome Res. 2006, 5, 1543–1554.
consisting of the same stationary phase. Furthermore, such a com-
[5] Gilar, M., Olivova, P., Daly, A. E., Gebler, J. C., J. Sep. Sci.
bination addresses compatibility issues, thus allowing straightfor-
2005, 28, 1694–1703.
ward interfacing in on-line 2D LC con guration, as well as direct [6] Schley, C., Altmeyer, M., Mller, R., Swart, R., Huber, C. G., J. Paola Donato , Francesco Cacciola , Paola Dugo 1, 2 , Luigi Mondello 1, 2
1
1
linkage to a mass spectrometer.
Proteome Res. 2006, 5, 2760–2768.
The results achieved so far hold promise for further optimization of [7] Bushey, M. M., Jorgenson, J. W., Anal. Chem. 1990, 62,
the technique, as a valuable tool to investigate the nature of native 978–984. Abstract:
and recombinant proteins of clinical relevance. [8] Liu, Z., Patterson, D. G., Lee, M. L., Anal. Chem. 1995, 67 This bottom-up approach relies on both the power of 2D-LC separation techniques, and the sensitivity of MS (ESI-IT-TOF) detection. After separa-
3840–3845. tion by RP-LC×RP-LC is performed at the peptide level, both PDA and MS 2D plots are obtained by means of a dedicated software, which further
[9] Gu, H., Huang, Y., Carr, P. W., J. Chromatogr. A 2011, 1218, allows qualitative and quantitative data analysis, as well as spectral comparison/subtraction. Tandem MS data obtained by collision-induced
64–73. dissociation (CID) of the peptides were used for database search for α-casein and dephosphorylated α-casein, with high sequence coverage.
Keywords: RP-LC×RP-LC–PDA–IT-TOF, tryptic digest, proteome analysis, protein isoforms

1. Introduction
1. Intr oduction resolution and peak capacity, more homogeneous distribution of
peptides elution in the separation window, robustness and easy
The analysis of complex biochemical systems, such as proteins and handling [5, 6] .
peptides isolated from tissues, cells, and body fluids has always rep-
resented a major task for analytical chemistry. A high demand is in This technical report describes the first comprehensive 2D LC system,
fact placed both on the power of separation techniques, given the in which the on-line coupling of RP-LC×RP-LC to MS (IT-TOF) is investi-
extremely high complexity of the proteome samples, and on the gated (Fig. 1). The two dimensions consisted both of a novel fused-
sensitivity of detection methods, to enable probing of low abundant core stationary phase specifically designed for peptide separation,
proteins or peptides. Different strategies have been developed at- interfaced through an electronically activated 2-position, ten-port
tempting to address the needs of modern proteomics, increase the valve. The performance of the system was assessed by means of tryp-
overall throughput of proteomics experiments, and facilitate re- tic mapping (protein unfolding, trypsin digestion, and reversed-phase
searchers to investigate into the complicated biological networks in chromatography of the peptide samples), followed by ESI MS charac-
which proteins are involved, at different levels. terization of α-casein and dephosphorylated α-casein (Fig. 2).
For many years, two-dimensional polyacrylamide gel electrophoresis
(2D-PAGE) followed by mass spectrometry (MS) has been the work-
horse in analytical proteomics, its major drawbacks consisting in
difficulty of automation, low accessibility of membrane-bound
proteins, problematic detection of proteins with large molecular
weight, high pI, strong hydrophobicity, or low abundance [1–4] . Over
the last decade, considerable effort has been put in the develop-
ment of ultra-high efficiency liquid chromatographic (LC) method-
ologies, pushing gel-free separation techniques to evolve beside the
more troublesome and tedious 2D-PAGE experimental designs. 2D
Fig. 8 Identi ed peptides in the 2D RP-LC×RP-LC plot of α-casein tryptic digest
separation set-ups based on RP-LC are characterized by superior

First Edition: December, 2015

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The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its Fig. 2 Plots: α-casein (top) and dephosphorylated α-casein (bottom)
accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the
use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject Fig. 1 LC×LC instrumentation with PDA and LCMS-IT-TOF detection tryptic digests
to change without notice.
www.shimadzu.com/an/ © Shimadzu Corporation, 2015 1 University of Messina, Italy
2 Chromaleont S.r.l.