Electrospray mass spectrometry

For ES investigations, the samples were introduced into the instrument using flow injection, where the sample is injected into a current of eluent, but without any separation device. A mixture of water and methanol (66:33) with one per cent acetic acid was used as eluent with a rate of 0.100 ml per minute. The samples were injected from glass vials (if necessary with glass inserts) using the autosampler. Unless otherwise indicated, ten microlitres sample was injected. Between each run, an equilibrium run of at least one minute was performed. Data registration started immediately after the sample injection as signalled by the auto injector device. Data were acquired in full ion scan mode for at least one minute, depending on flow rate, sample concentration and sample-tubing interactions. The instrument parameters applied are shown in table 2.1.

Table 2.1: Instrument parameters for ES and NANOES investigations of enzymatic reactions. IS: spray voltage, OR: orifice voltage, RNG: ring voltage; Q0: Q0 voltage; DF: deflector voltage.

	ES		NANOES
IS	5,300	V	5,200	V
OR	60	V	80	V
RNG	295	V	250	V
Q0	-10	V	-6	V
DF	-200	V	-200	V
Nebuliser flow	12		0
Curtain flow	10		5
Step size	0.10	amu	0.10	amu
Scan time	1	minute	--

Reactions were conducted in glass sample vials. The vials were loaded with the respective substrate or standard solution and placed inside the autosampler. After the start of the first equilibration run, the reaction was started by adding the enzyme to the sample. The data acquisition was controlled by the Sample Control software. Mass spectra were acquired for one minute. Subsequently, the data obtained were converted to a single averaged mass spectrum using Multiview (part of the Mass Chrom software). No peak selection on the total ion current was performed to increase the reliability and reproducibility and to allow a future automatation. The mass spectra were converted to plain text files and further processed using the self-developed kassandra perl script (described in 3.1.3) to obtain the relative intensities for each compound involved in the reaction. The concentrations were calculated as described in 3.1.3.1.