Peer review of: Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins (2021)

Reviewer #1 (Remarks to the Author): 

Review of NCOMMS-20-09835 

I am quite positive about this paper. The issue the authors tackle, of no one ever finding “the smoking 
gun” of formaldehyde crosslinking, in the form of the anticipated modified peptides or crosslinked 
peptide products, is one that has vexed me and vexed colleagues. Formaldehyde crosslinking is really 
used a LOT! FFPE tissue samples is one huge domain. In addition ChIP, ChIP-chip, ChIP-seq, Hi-C, and 
many other widely used techniques all rely upon formaldehyde crosslinking. It seemed obvious that if 
it crosslinks things, one should be able to see the crosslinked products by mass spec, yet somehow, in 
spite of so much work, those products have not been visible. It is almost embarrassing for the 
chemistry enterprise as a whole, to have such an important and widespread chemistry, using a 
molecule with only 4 atoms, to somehow have a mysterious and unknown chemical behavior that 
behaves in a way we do not understand and defies predictions. 

So, the authors have done a great job here, of using simple reasoning and straightforward methods to 
ferret out the origin of the mystery, and they provide both a simple and straightforward mechanism 
and an approach that will allow them and others to see the reaction products henceforth. It is a great 
service to the field, removing a cloak of darkness that has hung over an important and very widely 
used chemistry. 

Aside this global positivity, I have a few minor comments, mostly small grammatical items.. 

- Abstract line 29 IO suggest “The mass spectrometry data…” rather than “Our mass spectrometry 
data…” 
- Line 31. I find the term “surface modifications” a bit odd. I don’t feel like the chemistry is modifying 
a surface, it is modifying amino acids on a protein which are presumably exposed to solvent… I 
suggest “amino acid modifications” ? 
- Intro line 64 delete “a” before “commonly” 
- Line 124 no need for the hyphen in “undergoing” 
- Line 152 delete “is” before “dominates” 
- Line 175 insert “on” before “its efficiency” 
- Line 208 “restrains” is missssspelled (sic) 
- Line 219 insert the word “comprise” before “1.6%” 
- Sentence line 238-239 is unclear to me… I don’t follow the logic of “The mild reactivity…suggests 
that they underlie …toxic effects…” Need to spell out the logic of this thought I’m not following. 
- Line 301 “Fractions” not “Fraction” 
In Figure 5 there is an “e” missing panel A “Formaldehyd “ 

Also I am checking to see if the authors are confident in what they are saying in Panel B – “a 
nucleophilic attack by the imine on a nearby lysine sidechain” - this indicates that the imine is more 
nucleophilic than the lysine primary amine, which I find surprising as such primary amines are 
generally considered to be strong nucleophiles. A bit of googling turned up this “There is therefore less 
+ charge at the imine carbon atom which makes it less electrophilic; thus whilst imines do react with 
nucleophiles in a similar fashion to carbonyl groups, they tend to require strong nucleophiles and more 
forcing reaction conditions. “ 
Supporting the general idea that in this reaction the imine is the electrophile and the amine the 
nucleophile. If my chemistry building was not closed due to the Covid-19 pandemic I have a dozen 
colleagues who could answer this question for me in a millisecond.. however I am on my own, and so I 
just suggest the authors carefully consider their statement, it would be a shame to have such a nice 
paper include a possibly incorrect statement, especially in the most important figure that contains the 
major “take-home” message of the paper. 

A final point to consider relates to reversibility. 

The mechanism in Fig. 5 should show double arrows for all reactions to indicate reversibility, and then 
this supposition of reversibility should be mentioned/discussed in the text somewhere. All indications 
in the world are that formaldehyde is a reversible crosslinker, and therefore all steps of the 
mechanism are quite likely reversible. And there is no leaving group, just a concerted reaction that 
rearranges electrons, which is highly likely to be reversible. And the MS2 data presented provides 
strong evidence of reversibility. 



Reviewer #2 (Remarks to the Author): 

The manuscript from Tayri-Wilk, Slavin, and Zamel et al re-examines the characterization of 
formaldehyde crosslinks by mass spectrometry. The accepted mechanism, which is decades old, is 
that protein amines are crosslinked via a methylene bridge after initial Schiff base formation. The 
mechanism supposed here, 
is that two proximal Schiff bases undergo a cycloadditon to form a 1,3-diazetidine linkage. This is a 
highly ring strained 4-membered ring with two carbon atoms each bonded to two heteroatoms, and I 
find this structure implausible. 

However, overall the data presented here are actually fairly compelling. The idea of mass modification 
searching is common to look for linear modifications, but applying this strategy to scanning the 
crosslinker bridge mass is novel. The high-prevalence of +24 related modifications in the mod-
histogram is a solid piece of evidence, and this is supported by the evidence that after gas-phase 
collisional dissociation of the 
crosslinked peptides, the dissociation products are consistent with two linear peptide each modified by 
+12. Furthermore, the mappable crosslinks are consistent with known crystallographic structure and 
only form on structured proteins. 

FA is a highly attractive reagent to use for large-scale crosslinking studies due to its membrane 
permeability and long history of applications in a variety of cell and tissue fixation applications. The 
feasibility of FA-CLMS analysis where FA can be searched as a gas-phase labile linker analogous to 
DSSO is a rather tasty idea. 

As mentioned, I do have trouble swallowing the cyclic intermediate proposed. However, the 
manuscript should focus on the practical aspects of analyzing CLMS data using this approach, rather 
than getting bogged down in reaction mechanism. My suggestions below are oriented towards working 
out more basic aspects of this chemistry for the purpose of improving the robustness of the 
applications. 

1) The inability to find FA crosslinks is typically attributed to instability of the methylene bridged 
intermediates. The authors note that they keep their experimental preps below 40C to prevent 
decomposition. The addition of some experiments that explicitly characterized the chemical stability of 
FA crosslinks would be highly 
helpful to defining the general utility of this method. Particularly with respect to pH and temperature 
as these sample preparation and enrichment strategies used in proteomics, might for instance require 
high pH fractionation. 

2) The authors strategy for searching complex databases utilized a +24 Da variable modification with 
no residue specificity. This is inefficient and also likely to increase the error rate of this type of search. 
More experiments to define the amino acid specificity of FA crosslinking would be both useful and help 
with clarifying the mechanism. There are a number of papers by Kast group and others that have 
probed this extensively with linear peptides. I believe the Kast paper (ref 10) shows that at mild 
reaction conditions and short times, primarily Lys and Arg are modified and that other amino acids are 
only FA modified after days of reaction. Perusing the supplemental spectra here, there are cases 
where there are no K residues in the peptides and the pattern of +12 containing product ions (called 
B- and Y-ion here, see comment below) imply a modification on residues with no modifiable 
heteroatoms (I, L, V G, etc.). (eg P210L_HUMANACTB_HUMAN9229AVLIAESTQPIRAVFPSIVGRPR). I 
recommend searching the standard protein data with different residue specificity to try to establish the 
reactivity profile more clearly. 

3) The methods section does not mention the collision parameters for the HCD. Definitely add this, but 
also a study optimizing this should be added. In particular, is it possible at low energy to preferentially 
cleave the crosslink but leave the peptides largely intact for MS3 targeting. If the author have access 
to an ETD enabled instrument, I would also be interested in seeing whether the FA bridges are 
dissociated by electron transfer. 

minor - 

A) The authors mention that there is no direct evidence for observation of methylene bridged 
crosslinks. While I don’t disagree with their general conclusions, (ie model peptide based reactions are 
a poor way to study this), I don’t strictly speaking think this is true. For instance, ref 5, finds reaction 
between model peptide and glycine that are mediated by FA. Table I in that reference shows that a 
mass difference of +87 Da (eg Gly + 12Da) is the most common adduct. However, same table also 
finds +99 Da adducts which they attribute to multiple methylene bridges, but are also consistent with 
the mechanism here. 

B) Their search software annotates product ions that have a +12 Da modification as B- and Y- ions 
(upper case). I would avoid this, since this nomenclature is already well established for Glycopeptide 
fragmentation. 

C) For the mass modification scans - are these scanned as integer Dalton values? General practice is 
to model the average peptide distribution (“averagine”) and add a mass defect based on this. 

D) Authors should probably formally separate out searching for mis-annotated mono-isotopic masses 
and the crosslink search in the search engine. Searching for +24, +25, +26 is fairly awkward and 
could be handled by a neutral loss search for 1 or 2-Da.