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Native-MS Deconvolution Advanced Commands

  • Updated

Native mass spectrometry is a powerful method to study the interaction of protein complexes.  This technique is commonly coupled with size exclusion chromatography in biopharma to characterize biotherapeutic size heterogeneity, such as dimerization, aggregation, and clipping. Deconvolution of native mass spectra, however, often presents a challenge due to deconvolution over large mass ranges, fewer and lower charge states, and the presence of salt adducts.  
Intact Mass software offers a number of features to address the challenges of native mass spectrometry. This Insights Feature Focus covers the relevant advanced commands (text input to “Advanced configuration”).


Multimers

Deconvolution of native mass spectra of protein complexes, especially deconvolution of homo-multimers, poses a special problem: the same m/z peak may be shared by more than one mass. In particular, every m/z peak explained by a mass of m can also be explained by a mass of 2m.  Intact Mass v4.2 launched a new and improved method that puts more emphasis on smooth consecutive charging for each mass and less emphasis on parsimony, so that monomers and dimers, in fact, all homo- and hetero-multimers, will have accurate peak heights in the neutral mass spectrum. To use the new method, add the following advanced command:

[Intact]
ChargeEnvelopeFilterPower2=true

The new method not only improves deconvolution of multimers in native MS; it also improves deconvolution of low-abundance proteoforms in denaturing MS. For low signal-to-noise m/z peaks, the new method can improve Limit of Detection (LoD) more than 10-fold!  

(note: older advanced commandsMassCharge and CheckDoubleMass are now deprecated.)

The default command works well for spectra from denaturing conditions, in which the masses of interest have at least 5 consecutive abundant charge states and larger masses have more charge states than smaller masses. For native spectra in which masses have fewer charge states, the user should specify the filter (a symmetric sequence of numbers summing to 1.0) with another advanced command, ChargeEnvelopeWeights. For example, the following advanced command will direct the software to combine 3 consecutive charge states.

[Intact]
ChargeEnvelopeWeights=0.1, 0.8, 0.1

Linear Stepping for Wide Mass Ranges

With wide m/z and m deconvolution ranges, the follow advanced command improves deconvolution.

[Intact]
SteppingMethod=Linear
Resolution=30000

With stepping method set to linear, Intact Mass widens both the m/z and m spacings from left to right across spectra. Linear stepping better reflects the resolution of MS instruments, and can produce neutral mass spectra with isotope resolution at low m and smooth peaks at high m.

With SteppingMethod=Linear, the point spacing is not set by “Spacing (m/z)” and “Mass spacing” on the Advanced tab of the Compute menu, but rather it is set by theResolution. Resolution of 30000 or 35000 works well for most instruments and targets, but lower values may speed up processing for native MS on some instruments when the signal is at high m/z.
Protein Metrics’ parsimonious deconvolution allows deconvolution over a large mass range with fewer artifacts, especially half-mass artifacts.  Deconvoluted mass spectrum peaks marked with color dots (Lower Panel) in selection marquee mode are shown in the corresponding peaks in MS1 plots (Upper Panel) marked by the same-colored dots.
Point Spacing

By default, Intact Mass software resamples MS1 spectra at a fixed m/z spacing in order to simplify scan summing. The default point spacing of 0.005 works well for a wide variety of data, but this spacing may be overly fine for native mass spectrometry, leading to large file sizes.  The following advanced command changes the point spacing to 0.01. 

[Intact]
MzSampleSpacing=0.01

This advanced command must be set at project creation. Changing m/z sample spacing from 0.005 to 0.01 or an even larger value will reduce file sizes, both the .ntms project file and the (deletable) .cache file. A good rule of thumb is 5 sample points across an m/z peak, so that sample spacing of 0.04 is sufficient for peaks of width 0.2.
ample points across an m/z peak, so that sample spacing of 0.04 is sufficient for peaks of width 0.2.

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