Enhancing Top-Down Proteomics Of Brain Tissue With FAIMS Part 4

We also identified numerous fragments derived from non-canonical splice isoforms of tau protein known to be expressed in the human brain.83 Briefly, tau isoforms are defined by the number of N-terminal inserts due to alternative splicing of exon 2 and/or 3 (referred to as 0N, 1N, and 2N) and the number of microtubule-binding repeats due to alternative splicing of exon 10 (referred to as 3R or 4R).84 

Tau protein is an important protein in neuronal cells, which plays a very important role in maintaining the normal structure and function of neuronal cells. Studies have shown that tau protein is closely related to memory, and its abnormal expression can lead to cognitive decline and even cognitive impairment.

Scientists have done a lot of research on the relationship between tau protein and memory. According to research, tau protein can help regulate the stability of nerve fibers, thereby promoting information transmission and memory formation between neuronal cells. However, once the tau protein is abnormal, such as protein accumulation, it will lead to the breakage of nerve fibers and the death of a large number of neuronal cells, which will affect the formation and maintenance of memory and even lead to memory impairment. Therefore, maintaining the normal expression and function of tau protein is very important for maintaining the health of memory.

Recent studies have also found that the nutrients contained in some foods and beverages are also beneficial to maintaining the normal function of tau protein. For example, antioxidants, Omega-3 fatty acids, and caffeine-rich vegetables and fruits can help regulate the expression and function of tau protein, which helps improve memory and cognitive function.

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It is also worth noting that endogenous fragments of tau are a common finding in human brain tissue and cerebrospinal fluid, and differential fragmentation of tau may play a role in AD progression.85,86 

We observed several fragments that could unambiguously distinguish 0N and 1N tau (Figures 10A, S7, and S8). The fact that we observe high spectral counts for 0N (86 PrSMs) and 1N (91 PrSMs), but do not observe any PrSMs for 2N, is aligned with previous quantitative immunoblotting that found 0N and 1N to be the dominant forms and make ∼91% of total tau while 2N comprised only ∼9%.87 

About the unambiguous assignment of the microtubule-binding repeats, we observed spectra that could be assigned to both 3R (Figures 10A and S9) and 4R tau (Figures 10A and S10) as well. 

Parsimonious inference allowed us to conclude that brain tau is represented mostly by the mixture of 1N3R (Tau-B) followed by 0N4R (Tau-D) splice variants, which is in agreement with previous observations.87,88 

While other splice isoforms also had unique fragments, the low number of spectral matches (<4) did not allow us to confidently conclude about their existence.

Surprisingly, the N-termini of the predominant R-domain containing fragments are a continuation from the C-termini of the predominant N-domain containing fragments, or in other words, they appear to be linked fragments produced from a possible proteolytic cleavage event (referred to as site #1 in Figure 10A). Furthermore, the C-termini of the 3R and 4R domain-containing proteoforms share the same cleavage site (referred to as site #2 in Figure 10A), even though the sequences themselves are different due to splice variation of exon 10. 

Even more surprising, all of these cleavage sites lie within three well-described hexapeptide motifs.89–95 Figure 10B demonstrates all of the unique fragments we identified from our data sets that could be mapped to the 1N3R tau isoform, as well as the location of the cleavage sites. 

Cleavage site #1 lies within the second proline-rich region of tau, which divides the KVAVVR hexapeptide sequence and provides one of the strongest binding sites for microtubules (Figure 10B).89–91 Cleavage site #2 lies within the 3R and 4R hexapeptide motifs VQIVYK and VQIINK, which have been demonstrated to drive aggregation of tau (Figure 10B).92–95 

Interestingly, many of the fragments, as shown in Figure 10B, begin or end within the proximity of these two cleavage sites. Each of these hexapeptide motifs is known to form a β-structure, often in the form of β-hairpins which underlie the aggregation involved in various neurodegenerative diseases.89,93,96 Intriguingly, these observations highlight the possibility of a shared proteolytic degradation pathway among tau isoforms that is capable of disrupting their aggregation-seeding regions.25,85,86 

Lastly, we found numerous spectral matches for Aβ that could only be observed exclusively by using FAIMS. 

These Aβ proteoforms, which typically require special fractionation or handling techniques to improve recovery due to their hydrophobic and aggregation-prone properties,9,16,97,98 were observed intact using FAIMS within the −40 to −50 CV range. 

This includes the canonical Aβ1–42 and Aβ1–40 (Figure 11A, B, respectively), as well as several N-terminally truncated forms (specifically Aβ2–42 and Aβ4–42, Figure 11C, D). 

It is worth noting that Aβ proteoforms with various N- and C-terminal cleavages are notoriously difficult to identify in bottom-up analyses due to the extreme hydrophobicity of the tryptic products, which highlights an advantage of intact protein analysis. 

We believe that the FAIMS-TDP methodology offers a robust way for achieving intact identification of these Aβ proteoforms, allowing for facile determination of the different combinations of N and C-terminal cleavage products that exist.

CONCLUSIONS

We have described how nano-LC reverse-phase separation of a highly complex sample containing proteins, over a wide mass range, can benefit from the implementation of gasphase fractionation through FAIMS in the context of top-down mass spectrometry. 

FAIMS was demonstrated to impact the transmission of proteoforms by size and/or charge, reducing MS1 complexity and allowing greater depth of coverage of the proteome. 

FAIMS at a single CV (−50) enabled the identification of 1833 ± 17 unique proteoforms on average, more than double compared to without FAIMS (754 ± 35). The addition of FAIMS did not result in a deterioration of quantification reproducibility and remained near 20% RSD, which is comparable with label-free bottom-up approaches. 

Decreasing CV of FAIMS was noted to increase the molecular mass of ion species being transmitted to the instrument (median MW of ∼5 kDa at −50 CV vs ∼15 kDa at −20 CV), and modulation of CV was also observed to influence the transmission of a proteoform's charge state envelope differentially. 

We also defined optimal combinations of CVs that could produce the largest theoretical maximum of proteoforms/genes or proteome sequence coverage. Compared to the three "No FAIMS" data sets, external CV stepping at −50, −40, and −30 V could more than double the number of unique proteoforms, unique genes, and proteome sequence coverage. 

It is also worth pointing out that our work only explored a relatively small CV range which we felt best suited our TDP approach using a complex sample. Since FAIMS can easily be adapted to other methods, low-resolution MS1 analyses with Orbitrap analyzers may be able to improve on the identification of larger proteoforms within the CV range we tested, as well as beyond −20 V where larger proteoforms would likely be observed. 

It can also be envisioned that future mass analyzer instrumentation that improves on the acquisition of larger proteoforms and is compatible with FAIMS may benefit from exploring CVs below −20, extending into the positive voltage range. 

Our TDP workflow allowed us to identify and characterize unique proteoforms derived from genes with known roles in neurodegenerative diseases. 

This included the determination of the composition of an unknown mass shift present near the iron-binding domain of α-synuclein, which was used to pinpoint the locations of potential iron-binding domains in β- and γsynuclein as well. PARK7 was also found to be modified with a succinyl group on its active site Cys residue. 

We were able to unambiguously distinguish tau fragments corresponding to 0N, 1N, 3R, and 4R splice variant isoforms individually and describe new proteolytic cleavage sites located within or near several aggregation-seeding hexapeptide repeats. 

Finally, FAIMS enabled the identification of several intact Aβ proteoforms, including the aggregation-prone Aβ1–42, without the need for complex fractionation or purification techniques. 

In summary, we believe that the addition of FAIMS to discover TDP will provide a robust and reproducible method for increasing the proteome available for detection and characterization.

Supplementary Material

Refer to the Web version on PubMed Central for supplementary material.

ACKNOWLEDGMENTS

The authors would like to thank Tao Liu and Richard Smith for their advice and helpful discussions. 

This work was supported by U01 AG061356 (P.L.D.J.) and R01 AG015819 (D.A.B.). A portion of the research was performed using EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Biological and Environmental Research program. Graphical abstract and image assets within Figure 1 were created using Biorender.com.

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