PLOT
Porous Layer Open Tubular Columns (PLOT)
Because ESI-MS is concentration-sensitive rather than mass sensitive, greatly improved limits of detection in LC-MS have been obtained by using narrow-bore columns. The high back-pressure of packed capillary columns motivated the development of monolithic capillary columns (below), and further improvements have been obtained with ultranarrow bore columns having the stationary phase in a porous layer on an open tube.
Overview and References
Porous Layer Open Tubular Columns
Nanospray ESI-MS, at low nL/min flow rates, has enormous advantages over conventional ESI-MS: much better sensitivity and ionization efficiency, greatly reduced charge competition and ion suppression. Therefore, new LC/MS approaches utilizing ultranarrow-bore LC columns are invaluable for the analysis of limited sample amounts, for example, proteomic analysis of several thousand cells collected by laser capture microdissection (LCM). Recently, we developed a robust protocol for production of 10 um i.d. porous-layer open tubular (PLOT) columns (photo), utilizing polystyrene-divinylbenzene for the porous layer, and operating at flow rates of 20 nL/min (1). On-line 1D and 2D PLOT/MS platforms were developed which optimized the resolving power of the PLOT column and facilitated the handling of samples of a few microliters in volume (2,3). The new platform enabled the comprehensive characterization of the proteome of 15,000 metastatic breast cancer cells collected by LCM. In total, over 8000 peptides were identified, covering nearly 2400 unique proteins with a false discovery rate of 2.5% at the protein level.
Glycosylation plays a crucial role in many biological process, including antibody recognition, intracellular transportation, and cell-cell interactions. Consequently many clinical biomarkers and therapeutic targets are glycoproteins. Comprehensive glycan analysis has long been a challenge because of their complexity, low MS response, and high hydrophilicity. Analysis of glycans using LC/ESI/MS is promising, but would require (i) new chromatographic approaches to resolve such polar analytes effectively and (ii) minimization of the ion suppression of these compounds during ESI. Addressing these issues, we have developed a new approach for the preparation of PLOT columns for hydrophilic interaction chromatography (HILIC). The amine-HILIC PLOT columns have high resolving power for glycans at a flow rate of 20 nL/min. When coupled on-line with ESI-MS, the platform demonstrated good sensitivity in glycan analysis, where detection is usually problematic due to ion suppression. To illustrate the high separation efficiency and high sensitivity achieved, 28 N-glycans were confidently identified from 3 ng of ovalbumin PNGase F digest, in a single HILIC LC/MS analysis. High quality MS/MS spectra were obtained and manually interpreted to assign glycan compositions. In addition, nanoLC/MSn characterization of glycan structures was also achieved. (4)
1. Yue, G., Luo, Q., Zhang, J. , Wu, S., Karger, B. L. Anal. Chem. 2007, 79, 938-946.
2. Luo, Q., Yue, G., Valaskovic, G. A., Gu, Y., Wu, S., Karger, B. L. Anal. Chem. 2007, 79, 6174-6181.
3. Luo, Q., Gu, Y., Wu, S., Rejtar, T., Karger, B. L. Electrophoresis, 2008, 1604-1611.
4. Luo, Q., Rejtar, T., Wu, S., Karger, B.L. J. Chromatogr. A In press.