Abstract
<jats:p> The analysis of high molecular weight or heterogeneous macromolecules is a significant analytical challenge. This is especially the case in mass spectrometry, where heterogeneity results in unresolved signal in <jats:italic toggle="yes">m/z</jats:italic> from which charge, and thus mass, information cannot be discerned. Recent developments in mass spectrometry-based approaches overcome these challenges by the simultaneous detection of <jats:italic toggle="yes">m/z</jats:italic> and charge, and their expanded application has been aided by the development of commercial instrument platforms for transmitting large, heterogeneous molecules. However, the characterization of heterogeneous species that span a broad range in <jats:italic toggle="yes">m/z</jats:italic> , such as glycoproteins, polysaccharides, or polymers, is still particularly challenging due to <jats:italic toggle="yes">m/z</jats:italic> -bias that is induced by the conventional approach of tuning a mass spectrometer for optimal transmission over a narrow window in <jats:italic toggle="yes">m/z</jats:italic> . To assist MS users in understanding and characterizing this <jats:italic toggle="yes">m/z</jats:italic> -bias, the reader is guided through the entire ion path of an Orbitrap UHMR mass spectrometer and explained the purpose and effect of each component on ion transmission. On the basis of this knowledge, practical recommendations for instrument tuning are presented with the aim of mitigating <jats:italic toggle="yes">m/z</jats:italic> bias and standardizing mass spectrum data reporting. The main target audience of this tutorial are bio-, synthetic or analytical chemists with an interest in the characterization of heterogeneous molecules and mixtures, or scientists interested in understanding each of the factors that contribute to the generation of a mass spectrum on one of the most widely used commercial instrument platforms in the world. </jats:p>