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Top-Down and Middle-Down Measurements

Top-down MS refers to an analysis where the intact protein is introduced into the MS without in-solution enzymatic or chemical fragmentation (Figure 1).  Structural information is obtained from the fragmentation pattern of the intact protein generated in the MS; gas-phase fragmentation of proteins mainly relies on ECD and ETD and to a lesser extent CID.

Top-down MS is useful for the rapid characterization of small to medium sized proteins.  High resolution mass spectrometers are required for top-down analyses due to the large number of highly charged fragment ions produced.  As a rule of thumb, the size of the protein to be analyzed by top-down MS should be in the same order of magnitude as the resolution of the instrument, e.g. a Q-ToF instrument with a resolution of 10,000 should produce reasonable structural information for proteins up to 10,000 Da.  For higher molecular weight proteins a higher resolution instrument will be required, for example a Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR MS, resolution 106) or an Orbitrap MS (resolution 105).

Top-down MS measurements produce N-terminal fragments corresponding to the variable regions of the heavy chain and the light chain as the major species, making this a convenient method to confirm the sequence or identify modifications within these regions.  The variable regions determine the mAb activity, and the sequences of these regions are unique to the specific mAb, therefore, it is particularly beneficial to be able to rapidly characterize them.
Another major advantage of top-down MS is that little material is consumed, structural information (relating to the variable and terminal regions) is obtained in a short time, and little sample preparation is needed when combined with LC-MS and in-source fragmentation.

 

Figure 1: Top-down analysis.

Middle-down experiments can be used as an alternative to top-down experiments to provide greater structural resolution.  In a middle-down experiment, the protein is cleaved into a few large fragments by disulfide bond reduction or limited proteolysis in the same way as a middle-up experiment (Figure 2).  The fragments are then introduced into the mass spectrometer for MS/MS analysis.   

Middle-down experiments require some sample preparation (disulfide bond reduction or limited proteolysis), however, minimum sample concentration is required as these chemical reactions employ a large excess of reagent.  This makes middle-down experiments preferable over bottom-up for low concentration samples.  However, when the variable domain is the only area of interest for characterization, a top-down experiment is preferred due to the minimal sample preparation required.

 
Figure 2: Middle-down analysis.

There are two MS modes of operation for top- or middle-down experiments.  In the first mode, precursor ions are fragmented without isolation (in-source fragmentation).  This type of fragmentation potentially has higher dissociation efficiency because the protein is fragmented before it has time to fold into a compact structure, and higher sensitivity because ions of different charge states are fragmented simultaneously, however, interference from contaminants can lead to poor specificity.  In-source fragmentation is commonly used for top-down analysis of intact mAbs. The second mode is an MS/MS experiment in which the precursor ion of interest is isolated prior to fragmentation, resulting in high specificity but lower sensitivity.  Both modes are useful for middle-down experiments depending on the requirements for specificity or sensitivity; however, MS/MS experiments are preferred to obtain the maximum amount of sequence information.   

 

 

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