NanoLC

Pinkse, M.W., Uitto, P.M., Hilhorst, M.J., Ooms, B., Heck, A.J. (2004).. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal. Chem. 76, 3935-3943. 

Y. Shen, N. Tolic, C. Masselon, L. Pasa-Tolic, D. G. Camp, K. K. Hixson, R. Zhao, G. A. Anderson, and R. D. Smith. Ultrasensitive Proteomics Using High-Efficiency On-Line Micro-SPE-NanoLC-NanoESI MS and MS/MS. Anal. Chem., 76(2004) 144-154. 

Separation and detection techniques for antibacterials in food mainly focus on the use of LC coupled to MS or tandem MS. Nevertheless, recent studies have suggested capillary electrophoresis coupled to laser-induced fluorescence (LIE) as a way of improving sensitivity [49], HRLC coupled to microTOF-ESI-MS as a highly selective, sensitive, and quick screening method for 100 veterinary drugs in fish, meat, and egg samples [195], and nanoscale LC coupled to UV or ion trap MS, with LODs in the range 0.01-0.51 pg/L (nanoLC-MS) and the possibility that even lower limits could be achieved by using triple quadrupole MS [59]. 

Schlosser, A., Vanselow, J.T. and Kramer, A. (2005) Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and NanoLC-MS/MS analysis. Analytical Chemistry, 77, 5243-5250. 

FIGURE 3.4 (A) The NanoLC 2-D system by Eksigent Technologies, used in our laboratory for aHPLC... 

Shen, Y., Tolic, N., Masselon, C., Pasa-Tohc, L., Camp, D. G., Hixson, K. K., Zhao, R., Anderson, G. A., and Smith, R. D., Ultrasensitive proteomics using high-efficiency on-line micro-SPE-NanoLC-NanoESI MS and MS/MS, Analytical Chemistry 76(1), 144-154, 2004. 

Reverse-phase columns with a gradient elution in combination with UV-Vis spectrophotometers using photodiode-array (PDA) (Fig. 1.6) and spectrofiuorimeters are common devices employed in this technique. In a lesser extent, MS, tandem mass spectrometry (MS-MS), and nano liquid chromatography-electrospray ionization-quadrupole time-of-flight tandem mass spectrometry (nanoLC-nanoESI-Q-qTOF-MS-MS) has been used as detection system. This instrumentation has been mainly used in the analysis of dyes and proteinaceous media, and in some extent, in the analysis of drying oils and terpenoid varnishes [47,48],... 

Developments in mass spectrometry technology, together with the availability of extensive DNA and protein sequence databases and software tools for data mining, has made possible rapid and sensitive mass spectrometry-based procedures for protein identification. Two basic types of mass spectrometers are commonly used for this purpose Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) and electrospray ionization (ESI)-MS. MALDI-TOF instruments are now quite common in biochemistry laboratories and are very simple to use, requiring no special training. ESI instruments, usually coupled to capillary/nanoLC systems, are more complex and require expert operators. We will therefore focus on the use of MALDI-... 

Use these solutions directly for MALDI-MS and nanoLC-ESI-MS or desalt them... 

MW—molecular weight synonym of the molecular mass (abbreviated M)—is the mass of one molecule NALT—nose-associated lymphoid tissue nanoLC-MSMS—nanoliquid chromatography-tandem MS NCF—neutrophil chemotactic factors NFA—nuclear factor of activated T-lymphocytes NF-kappaB—nuclear factor kappa B NK—cells natural killer cells NMR—nuclear magnetic resonance... 

Fourier transform Ion cyclotron resonance ESI, APCl, MALDl, E1,C1 0.1-5 >1,500,000 -250,000 Very high resolution, high-accuracy m/z analysis LC/MS, nanoLC/MS, and MALDl... 

The complementarity of all the data obtained in this strategy (LC-MALDI-MS and nanoLC-MS/MS) allowed to increase significantly the coverage percentage for all the gel slices. Figure 3 summarizes this strategy and technical details are presented below. 

Table 1 reports the different proteins identified during the nanoLC-MS/MS analysis of a single ID gel slice. Peptides obtained from the nanoLC-MS/MS and... 

Figure 5. Summary of the strategy applied for a better sequence coverage of the identified proteins in ID gel. The first step is the analysis of the raw peptide mixture by MALDI. The step 2 corresponds to the nanoLC-MS/MS analysis of the peptide digests, and the step 3 corresponds to the off-line fractionation of the peptide digest and MALDI-MS analysis. The combination of these approaches allow a better coverage of the different proteins identified.

Moreover, we have determined the false positive rate for this approach. Many tryptic peptides originated from different proteins can be attributed to a single mass (e.g. HQHPLQCVMEK 1364.63 Da and EADFINCVIWR 1364.65 Da AM < 20 ppm). Thereby false positive identification may occur. To evaluate the false positive rate, we have selected three common proteins which were not identified during the nanoLC-MS/MS analysis. These proteins (tubulin, actin and myosin) were digested in-silico, and the generated mass lists were compared to the LC-MALDI-MS peaklist. A total of only five masses were attributed to the three... 

Table 1. Sununary of the proteins identified by nanoLC-MS/MS from a single 1D gel slice, and the increase of the coverage using LC-MALDI-MS strategy...

Accession number Protein Peptides identified by nanoLC-MS/MS Coverage (%) Peptides identified by LC-MALDl Combination MALDI/ESl ... 

To conclude, we have developed a strategy which combines LC-MALDI-MS and nanoLC-MS/MS in order to identify proteins originating from ID gel with a high coverage compatible with plasma membrane study (CD98, CD71, CD44). 

In order to validate the hypothesis that microparticles are structures enriched in plasma membrane proteins, we have analysed the proteome composition of different microparticle preparations obtained from a T-Lymphocytic cell line. Such microparticles can be produced and enriched in vitro by mitogenic activation (PHA) or apoptosis induction (Act Dl, TNFo ) for instance. Microparticles protein mixtures obtained from the two stimulations were separated independently on a ID gel. The different gels were cut each 2 mm and the different slides were digested and analyzed by nanoLC-MS/MS. 

The nano-electrospray (nanoES) source is essentially a miniaturized version of the ES source. This technique allows very small amounts of sample to be ionized efficiently at nanoliters per minute flow rates and it involves loading sample volumes of 1-2 pi into a gold-coated capillary needle, which is introduced to the ion source. Alternatively for on-line nanoLC-MS experiments the end of the nanoLC column serves as the nanospray needle. The nanoES source disperses the liquid analyte entirely by electrostatic means [27] and does not require assistance such as solvent pumps or nebulizing gas. This improves sample desolvation and ionization and sample loading can be made to last 30 minutes or more. Also, the creation of nanodroplets means a high surface area to volume ratio allowing the efficient use of the sample without losses. Additionally, the introduction of the Z-spray ion source on some instruments has enabled an increase in sensitivity. In a Z-spray ion source, the analyte ions follow a Z-shaped trajectory between the inlet tube to the final skimmer which differs from the linear trajectory of a conventional inlet. This allows ions to be diverted from neutral molecules such as solvents and buffers, resulting in enhanced sensitivity. 

NanoLC-ESI-MS of tryptically digested human IgG. Extracted chromatograms for the two main diagnostic glycan ions, HexHexNAc m/z 366 upper panei) and HexNAc m/z 204 lower panel) are shown. The chromatograms of both ions indicate that most of the glycopeptides elute between 10 and 18 minutes... 

NanoLC-ESI-MS of tryptically digested human IgG. The extracted chromatogram of the ion at m/z 1479 shows a signal at 15 minutes (upper panel). The lower panel shows the summation of all spectra acquired between 14 and 16 min. The resulting MS spectrum demonstrates the presence of several glycoforms of the peptide EEQYNSTYR. Monosaccharide symbols are shown in O Table 1... 

Magnified views of the nanoLC-ESI-MS/MS of the glycopeptide signal at m/z 13982. The b- and y-ions were used to sequence the peptide (summarised in top panel)... 

As highlighted in the reviews from Li et al. [71] and Hummon et al. [41], in addition to the mollusk single-cell models, MALDI-MS associated or not to nanoLC and on-line LC-ESI-MS/MS has been successfully applied to a wide variety of animal species from invertebrates to vertebrates. 

As ESI works on a continuous flow of liquid, it has quickly been coupled to LC or other liquid-phase separation techniques as an alternative to optical detection.15 Mass spectrometry gives more information on the eluted compound, and the resulting hyphenated technique enables one to decrease the complexity of samples before their analysis by MS. High performance liquid chromatography (HPLC) is coupled to conventional ESI-MS while nanoLC is connected to nanoESI-MS for a better match in the flow-rate values. 

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