posted on 2023-01-11, 17:43authored byWendong Ma, Irene Ling Ang, Kate M.K. Lei, Melody Man Ting Lam, Pengwei Zhang, Terence Chuen Wai Poon
Emerging
evidence suggests that advanced glycation end-products
(AGEs) such as Nε-(carboxymethyl)lysine
(CML) and Nε-(carboxymethyl)lysine
(CEL) may play important roles in certain human diseases. Reliable
analytical methods are needed for their characterizations and measurements.
Pitfalls have been reported for applications of LC–MS/MS to
identify various types of post-translational modifications, but not
yet for the case of AGEs. Here, we showed that in the absence of manual
inspection, cysteine alkylation with 2-iodoacetamide (IAA) can result
in false-positive/ambiguous identifications of CML >20%. They were
attributed to offsite alkylation together with incorrect monoisotopic
peak assignment (pitfall 1) or together with deamidation (pitfall
2). For pitfall 1, false-positive identifications can be alleviated
using a peptide mass error tolerance ≤5 ppm during the database
search. Pitfall 2 results in ambiguous modification assignments, which
may be overcome by using other alkylation reagents. According to calculations
of theoretical mass shifts, the use of other common alkylation reagents
(iodoacetic acid, 2-chloroacetamide, and acrylamide) should face similar
pitfalls. The use of acrylamide can result in false-positive identifications
of CEL instead of CML. Subsequently, we showed that compared to IAA,
the use of N-isopropylacrylamide (NIPAM) as an alkylation
reagent achieved similar levels of proteome coverage, while reducing
the offsite alkylation reactions at lysine by more than five times.
Furthermore, false-positive/ambiguous identifications of CML due to
the two types of pitfalls were absent when using NIPAM. NIPAM alkylation
results in a unique mass shift that allows reliable identifications
of CML and most likely other AGEs, such as CEL.