High proteoform count

[Sam Lord]

Hello,

Our lab has recently started top down proteomics and have began using the NRDTP standards (Ubiquitin, Myoglobin, Carnbonic Anhydrase). We are running on an LC-MS system and we see three nice intense peaks representing the full length proteoform. 

What we found surprising is that when we search with TopPIC we many (sometimes hundreds) of proteforms for each of the 3 proteins scattered across the gradient. What is strange is that this happens with or without in source fragmentation. We are not searching for lots of PTMs although we do reduce the numbers when we filter out unexpected mass shifts.

Do you have any explanation as to why we can see so many proteoforms and is this often seen with TopPIC analysis or perhaps are filtering.

I have attached an example of all the different proteoforms we see in terms of sequence length in myoglobin (we see even more for carbonic anydrase).

Many thanks

Sam

[Liu, Xiaowen (Kevin)]

Hi, Sam, 

Truncated proteoforms are commonly detected in top-down MS. They may be present in the original sample or generated during sample preparation, separation, or MS analysis. The following paper provides information on truncated proteoforms identified in other top-down MS datasets.

https://www.biorxiv.org/content/10.64898/2026.02.20.707032v2

Best, 

Xiaowen (Kevin)

From: 'Sam Lord' via TopPIC <top-dow...@googlegroups.com>
Date: Friday, September 18, 2026 at 3:20 AM
To: TopPIC <top-dow...@googlegroups.com>
Subject: [ms-align-plus-pipeline] High proteoform count

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