Ion mobility spectrometry-mass spectrometry (IMS-MS) determines momentum transfer cross sections of ions to elucidate their structures. Recent IMS methods employ electrodynamic fields or nonstationary buffer gases to separate ions. These methods require a calibration procedure to determine ion mobilities from the experimental data. This applies in particular to trapped IMS (TIMS), a novel IMS method with reported high resolving powers. Here, we report the first systematic assessment of the accuracy and the limitations of mobility calibration in TIMS. Our data show that the currently used TIMS calibration approach reproduces drift tube mobilities to approximately 1% (95th percentile). Furthermore, we develop a transferable and sample-independent calibration procedure for TIMS. The central aspects of our approach are (1) a calibration function derived from a solution to the Boltzmann transport equation and (2) calibration constants based on a Taylor expansion of instrument properties (TEIP). The key advantage of our calibration approach over current ones is its transferability: one equation and one set of parameters are sufficient to calibrate ion mobilities for various instrument settings, compound classes, or charge states. Our approach is transferable over time and sufficiently accurate (∼1-2%) for structure-elucidation purposes. While we develop our calibration procedure specifically for TIMS, the approach we take is generic in nature and can be applied to other IMS systems.

Metrics & MoreArticle Recommendations I on mobility spectrometry-mass spectrometry offers the potential to reveal structures of biological molecules not amenable to established biophysical methods. It is, however, still controversial if ion mobility truly reveals biologically relevant structures. This is because the measurement takes place in the gas phase, but it is not known for how long the native structure survives in this environment. 1 Ion mobility spectrometry is thus a nonequilibrium method 2 and successful only to the extent that native structures are metastable within the measurement time scale. 3,4 Ergo, the critical question is how much "heat" do ions take up from electric fields in an ion mobility spectrometer?In ref 5, Morsa et al. investigated ion heating in a trapped ion mobility spectrometer (TIMS). The authors claim that "Using the lowest possible transmission voltages [...the authors] obtained vibrational effective temperatures T eff,Vib of 512 K" for a thermometer ion which accounts for a "high [axial electric field] E/N above the low-field limit", from which the authors conclude that "the ion transport regimes in traditional [drift-tube ion mobility spectrometry] and modern [...] TIMS instrumentations differ therefore advising caution when comparing data obtained on the different platforms."If true, these claims made in ref 5 would have far-reaching, adverse implications on the use of TIMS for structural studies: if the ion separation process in TIMS truly heated molecules to ∼500 K, then ion mobilities would be unreliable and biological molecules would structurally denature in TIMS.Indeed, ref 5 expresses such concerns by "questioning the adequacy of the strategy [of keeping the ions stationary employed in TIMS to improve the mobility resolving power] for native mass spectrometry applications targeting fragile analytes associated with low activation energy barrier E a such as small molecules or noncovalent complexes."These claims, however, stand in stark contrast to experiences made by us and others that ion mobilities obtained from TIMS measurements are accurate 6,7 and, when carefully tuned, spectra from TIMS and tandem-TIMS resemble "soft" drifttube spectra for fragile noncovalent peptide assemblies, 8 monomeric proteins, 2,9−11 and even indicate solvent to be trapped inside protein assemblies. 12 Here, we clarify (a) that the ion heating to ∼500 K reported in ref 5 is not intrinsic to TIMS and (b) that TIMS enables native mass spectrometry applications:1. When discussing the low-field limit and vibrational ion heating in ion mobility spectrometry, all forces that

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Carbohydrates play important roles in biological processes, but their identification remains a significant analytical problem. While mass spectrometry has increasingly enabled the elucidation of carbohydrates, current approaches are limited in their abilities to differentiate isomeric carbohydrates when these are not separated prior to tandem−mass spectrometry analysis. This analytical challenge takes on increased relevance because of the pervasive presence of isomeric carbohydrates in biological systems. Here, we demonstrate that TIMS 2 −MS 2 workflows enabled by tandem-trapped ion mobility spectrometry−mass spectrometry (tTIMS/MS) provide a general approach to differentiate isomeric, nonseparated carbohydrates. Our analysis shows that (1) cross sections measured by TIMS are sufficiently precise and robust for ion identification; (2) fragment ion cross sections from TIMS 2 analysis can be analytically exploited to identify carbohydrate precursors even if the precursor ions are not separated by TIMS; (3) low-abundant fragment ions can be exploited to identify carbohydrate precursors even if the precursor ions are not separated by IMS. (4) MS 2 analysis of fragment ions produced by TIMS 2 can be used to validate and/or further characterize carbohydrate structures. Taken together, our analysis underlines the opportunities that tandem-ion mobility spectrometry/MS methods offer for the characterization of mixtures of isomeric carbohydrates.

Human B cell adaptor for phosphoinositide 3-kinase (BCAP) is identified as an adaptor protein expressed in B cells and plays a critical immunomodulatory role in B cell receptor signaling and humoral immune response. In the current study, a homolog of BCAP (Lja-BCAP) was identified in Lampetra japonica. The open reading frame of Lja-BCAP contains 2181bp nucleotides and encodes a protein of 726 amino acids. After being stimulated by mixed bacteria, the mRNA and protein expression levels of Lja-BCAP and the activation levels of tyrosine kinases increased significantly in peripheral blood lymphocytes, gills and supraneural myeloid bodies, respectively. However, after the knockdown of Lja-BCAP by RNAi in vivo, the activation of tyrosine kinases was inhibited in the above tissues, which indicated that Lja-BCAP participated in the anti-bacterial immune response of lampreys. After lipopolysaccharide (LPS) stimulation, the expression of Lja-BCAP in peripheral blood lymphocytes, gills and supraneural myeloid bodies were significantly up-regulated 2.5, 2.2, and 11.1 times (p < 0.05) compared to the control group, respectively; while after phytohemagglutinin (PHA) stimulation, the up-regulation of Lja-BCAP was only detected in peripheral blood lymphocytes. The above results show that Lja-BCAP mainly participates in the LPS-mediated immune response of lampreys.