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cLC-MALDI-MS

While several combinations of cLC-MALDI-MS have been reported, a system recently developed at the Genomics Institute of the Novartis Research Foundation is indicative of the state-of-the-art.1-4-1 As illustrated in Fig. 1, the outlet of the cLC column is fixed over the target plate mounted on a motor-driven translation stage, allowing fractions to be collected at discrete locations on the plate. To control the deposition of a fraction, droplets formed on the column outlet are pulled down to the plate by application of a voltage pulse. Novel use of voltage to dislodge droplets allows complete and automated control of sample deposition and, because the column is not in contact with the plate surface, opportunities for cross-contamination and damage to the column or plate are minimized. The target plate consists of hydrophobic surface punctuated with an array of 400-mm-diameter wells containing a hydrophilic surface coating.[5] The use of a prestructured surface prevents a deposited fraction from spreading on the surface and confines it to the predetermined area. In addition, it allows for fairly large samples to be concentrated onto a small area because large droplets adhere to the hydrophilic spot and are confined to the spot as they evaporate. The small sample area allows the laser to irradiate the entire spot, eliminating the ''sweet spot'' effect. Matrix may either be added postcolumn via a Y-connector or precoated onto the target plate in a multistep deposition, depending on the properties of the matrix. This prototype instrument utilizes up to four cLC columns (300-mm i.d., 15 cm long) in an array. Because chromatography separations can take 10-100 min, the use of the array can substantially increase the throughput of analysis.

The performance of this instrument was demonstrated by analysis of a tryptic digest of bovine serum albumin using a Fourier-transform ion cyclotron resonance (FT-ICR) instrument as the MS. A sample of the three-dimensional data output with separation time, m/z, and signal intensity as the dimensions is illustrated in Fig. 2.

Fig. 1 Automatic deposition of effluent from cLC directly down to a MALDI target plate. A) The automated off-line system for cLC-MALDI. B) Deposition of the effluent from the LC column down to the target plate by application of a negative voltage. (From Ref. [4].)

Fig. 2 3-D plot of LC/MALDI-MS analysis of 200 fmol BSA tryptic digest with elution profiles for two peptides (D363-K375 and L274-K285). 20 pL of a 10 fmol/mL digest solution was injected each onto four parallel columns. The effluent was deposited every 10 sec onto the target plate. A sequence coverage for BSA of 78% was obtained. (From Ref. [4].) (View this art in color at www.dekker.com.)

Fig. 2 3-D plot of LC/MALDI-MS analysis of 200 fmol BSA tryptic digest with elution profiles for two peptides (D363-K375 and L274-K285). 20 pL of a 10 fmol/mL digest solution was injected each onto four parallel columns. The effluent was deposited every 10 sec onto the target plate. A sequence coverage for BSA of 78% was obtained. (From Ref. [4].) (View this art in color at www.dekker.com.)

The detection limit for the instrument, when coupled to a time-of-flight mass spectrometer (TOF-MS), was better than 50 attomol. The instrument could collect up to 384 fractions from 4 columns, or 1536 fractions from a single column. This large number of fractions would make it possible for operators to obtain multiple fractions for all resolved analytes, because the peak capacity of an LC column, i.e., the maximum number of resolvable components, is typically ~ 200.

Off-line coupling of cLC-MALDI-MS may be achieved in other ways as well. For example, the effluent can also be deposited as a continuous trace on the target instead of discrete spots.[6] In this case, the column effluent passes through a heated nebulizer that sprays the effluent directly onto the target plate. The continuous trace deposition method allows the full chromatographic peak capacity to be retained.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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