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            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                                    resolution  and peak capacity,  more homogeneous distribution  of
                                                                                                                                   1. Intr
                                                                                                                                           oduction
                                                                                                                                                                                      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.                                                                                   1
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