Native Mass Spectrometry and Collision-Induced Unfolding of Laser-Ablated Proteins

Abstract

Infrared laser ablation sample transfer (LAST) was used to collect samples from solid surfaces for mass spectrometry under native spray conditions. Native mass spectrometry was utilized to probe the charge states and collision-induced unfolding (CIU) characteristics of bovine serum albumin (BSA), bovine hemoglobin (BHb), and jack-bean concanavalin A (ConA) via direct injection electrospray, after liquid extraction surface sampling, and after LAST. Each protein was deposited from solution on solid surfaces and laser-ablated for off-line analysis or sampled for online analysis. It was found that the protein ion gasphase charge-state distributions were comparable for direct infusion, liquid extraction, and laser ablation experiments. Moreover, calculated average collision cross section (CCS) values from direct injection, liquid extraction, and laser ablation experiments were consistent with previously reported literature values. Additionally, an equivalent number of mobility features and conformational turnovers were identified from unfolding pathways from all three methods for all charge states of each protein analyzed in this work. The presented work suggests that laser ablation yields intact proteins (BSA, BHb, and ConA), is compatible with native mass spectrometry, and could be suitable for spatially resolved interrogation of unfolding pathways of proteins.

Collision-Induced Unfolding, Tandem MS, Bottom-up Proteomics, and Interactomics for Identification of Protein Complexes in Native Surface Mass Spectrometry

“…In addition to bottom-up, NSMS, and tandem MS results, we utilized collision cross-section measurements to assess the match between measured CCS N2 values (peak centroid ± 2s) and those expected for the assigned proteins. 39 The calculated CCS N2 values for the ADH 4 charge states z range = +25−23 agree with those from conventional native nESI-MS reports (Supporting Figure S6) to within 3% of published values for these categories. 81,82 Although there are no prior native IM-MS reports for HRI1, AIM2, or HRI1-AIM2 heterodimer proteins, the measured CCS N2 values of 4000 ± 450 Å 2 , 3900 ± 300 Å 2 , and 3850 ± 325 Å 2 for charge-states +15, +14, and +13 of the HRI1-AIM2 heterodimer are reasonable values for a protein with a molecular weight of 54.8 kDa.…”

“…These three calculated CCS N2 values (Supporting Figure S15A−C) for CSA are within three percent of the reported literature values for these categories from purified substrates. 109 Similarly, for CCS calculations of the alpha-enolase dimer, three charge states +19, +18, and +17 were characterized, which yielded CCS values of 5400 ± 400 Å 2 , 5300 ± 325 Å 2 , and 5200 ± 275 Å 2 , respectively (Supporting Figure S15D To further solidify the stoichiometry assignments of the observed protein complexes, we performed a "charge multiplexed" 22,39,128 CIU analysis of the most abundant charge states of CSA, AENL 2 , and LDHA 4 (Figure 6). The unfolding of CSA was used as a reference calibrant to assess the quality of CIU plots and RMSD-based precision of multiplicate runs (see Supporting Figure S15) as its CIU has been reported earlier.…”

“…Additional details and operational principles of the components utilized to design, construct, manufacture, and keep the surface probe nESI assembly clean have been described previously. 39 The proteomics workflow for integrating NSMS, MS/MS, bottom-up, and ion mobility data is presented in Scheme 1. First, the sample was extracted in situ (A, Scheme 1) and split into two portions for parallel native mass spectrometry (B−D) and bottom-up proteomics (E−G) analyses.…”

“…37,38 We recently demonstrated that laser ablation is compatible with NSMS and IMS to probe surface sampled proteins' collision cross sections and unfolding patterns. 39 However, currently available sampling techniques are unsuitable for depth profiling and interrogating subsurface features under native MS conditions. Thus, the existing in situ characterization capabilities have not been directly translated to ex vivo or in vivo NSMS or ion mobility for protein identification.…”

“…This work presents a multiprong method for NSMS that utilizes in situ native nESI-IM-MS with collision-induced activation-unfolding, 39 tandem MS, and bottom-up proteomics 74 to identify proteins and protein complexes. This multiprong workflow is enabled by using a custom liquid extraction surface sampling device to withdraw proteins for NSMS, tandem MS, protein unfolding, and bottom-up experiments for proteomics screening from a single sampling event.…”