{"id":4672,"date":"2019-03-25T18:04:00","date_gmt":"2019-03-25T18:04:00","guid":{"rendered":"https:\/\/www.waters.com\/blog\/sample-prep-before-lc-ms-quantification-of-peptides-and-proteins\/"},"modified":"2022-07-28T03:10:38","modified_gmt":"2022-07-28T03:10:38","slug":"sample-prep-before-lc-ms-quantification-of-peptides-and-proteins","status":"publish","type":"post","link":"https:\/\/www.waters.com\/blog\/sample-prep-before-lc-ms-quantification-of-peptides-and-proteins\/","title":{"rendered":"Sample Prep before LC-MS Quantification of Peptides and Proteins"},"content":{"rendered":"

Confronting the challenge of sensitivity and sample loss<\/strong><\/h1>\n

As a relative newcomer to peptide and protein quantification coming from the small molecule world, I have learned that I need to think differently, and more creatively, to meet the challenges of these molecules. \u00a0As scientists with assay sensitivity goals, we sometimes have to take extra steps to ensure that we not only meet this<\/em> goal, but we also ensure reproducibility and overall robustness of the methods we develop.\u00a0 As I think about this challenge, I find it helpful to think of the workflow from the perspective of my sample.<\/p>\n

\"\"In part 1<\/u><\/a> of this blog series, Caitlin Dunning highlighted how easy it is to lose your sample\u00a0during\u00a0sample handling and discussed the consequences of doing nothing!<\/p>\n

Moon Jung, in part 2<\/u><\/a>, discussed non-specific binding, the major cause of sample loss, and the pros and cons of using traditional approaches such as blocking agents and carrier protein to minimize sample loss.<\/p>\n

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In this, part 3 of our blog series, I am going to explain why your choice of sample container is so important in minimizing non-specific binding and making sure you don\u2019t lose your precious sample. It is a bit like choosing the right hotel room for your vacation or holiday!<\/p>\n

Is there such a thing as a “peptide hotel”?\u00a0 If there is, how do I choose the right hotel?<\/strong><\/p>\n

Imagine you\u2019ve done the hard work and prepared your sample.\u00a0 It is nice and clean and concentrated: free of troublesome matrix interference and unfriendly column \/ mass spectrometer contaminants. Now it is going to sit in an LC-MS autosampler for a few hours, or even 2 – 3 days if a big run is planned over the weekend.<\/p>\n

Should I be worried about sample loss? Will I lose my peptide to non-specific binding in the sample plate or vial?<\/p><\/blockquote>\n

Careful selection of a sample container designed to reduce non-specific binding can help.<\/em><\/p>\n

Where can I find sample containers that have a low binding advantage, but are also optimal for the autosampler of the LC-MS system in both format and purpose?\u00a0 Do such containers even exist today?\u00a0 What I mean by this is, with total available sample volumes decreasing more and more each day, a vial or plate designed with low residual sample volumes is essential.<\/p>\n

This leads me to my final question.\u00a0 Does the sample concentration make a difference in non-specific binding?\u00a0 As Caitlin Dunning mentions in Part 1<\/u> \u201cWhere did my sample go?\u201d\u00a0 NSB doesn\u2019t just impact our ability to reach our sensitivity targets at lower concentrations.\u00a0 It can also lead to problems in variability.<\/p>\n

When we compare a standard polypropylene container to a container with a high performance surface designed to reduce NSB, we see a large difference in sensitivity for 1 ng\/mL of glucagon.\u00a0 However, we also see a difference when doing the sample comparison with 100 ng\/mL of bovine insulin!\u00a0 The rate of loss may vary depending on the amount of time the sample sits in the container, leading to variability even at higher concentrations.<\/p>\n

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Chromatographic peaks of challenging peptides after each peptide solution was stored in respective containers for 24 hours at 4 \u00b0C.<\/figcaption><\/figure>\n

Challenge Accepted<\/strong><\/p>\n

Careful consideration needs to be taken with peptide and protein quantification, especially compared to small molecule analysis.<\/p>\n