There's a wealth of information on LDI MS  for low m/z analysis out on the web that can be easily found using search engines.

 Please see the links below for reference:



A recent and an excellent review by Prof. Dietric Volmer from Saarland University on small molecule quantitation using mass spectrometry imaging: “Quantification of low molecular weight compounds by MALDI imaging mass spectrometry – A tutorial review”, Biochim. Biophys. Acta. 2017, 1865(7):726-739. doi: 10.1016/j.bbapap.2016.12.011

 Another nice review by Prof. Roger Giese: “Recommendations for quantitative analysis of small molecules by matrix-assisted laser desorption ionization mass spectrometry”, J. Chromatogr. A. 2017, 1486, 35-41; http://doi.org/10.1016/j.chroma.2017.01.040

Here's a summary of pointers that the Panchagnula group put together - sort of an initiation process for the new members in our group and beginners of mass spectrometry (credits: Nivedita, Deepika & Ajeet):

1. Always do a bit of homework and literature search before attempting any sample analysis. Know beforehand what kind of results one should expect. There are numerous examples already reported one can look up. An important thing about research is to be able to network and find help. Our group has benefitted by interactions, conversations and helpful exchanges from numerous stalwarts of mass spectrometry.

 2. Importantly, make sure that the matrix being used is the most preferred one for a particular analysis (and matrices are different for small molecules, peptides, proteins, oligos, polymers). Also, the concentration (5mg/ml? 10mg/mL?), premixing or prespotting, on top or below the sample - these are all considerations one should explore and have a handle. How does one dry the plate?

3.  Find out the concentration range needed for the particular type of analysis and if necessary incorporate a dilution or concentration step for the samples. More is not necessarily good in highly sensitive techniques such as mass spectrometry. Understand the concepts of S/N, noise, accurate mass, sensitivity, resolution and other basic MS terminology.

4.  Always have the reference standards ready. This is especially important in a resource limited geographical environment where even normal reference standards can sometimes take months to arrive after placing an order (let’s not even talk about labelled standards, embargos, customs clearances and costs!). In our group, we benefitted by talking to the synthetic chemists and narrowed down some standards that can be (relatively) easily procured. These include drugs (verapamil for example) and other precursors for various synthetic processes (triazines are our favorites). These can be handy when performing low m/z analysis in addition to synthetic peptides and protein standards normally used.

5.  Periodically check the inventory and status of the standards, matrices, the desiccators where these are stored and take a count of the plates to make sure you don't have anything missing. Standards and matrices should be aliquoted into usable portions and stored in the freezers. Matrix solutions are best prepared fresh before using.

6.  Pay attention to the target plate cleaning protocols and ensure that the plate is sufficiently cleaned to avoid carryover effects. Instrument manufacturers might have different target plates – so do check the manufacturer’s manuals and specific instructions.

7.  Check whether your samples have dried or not completely before inserting in the instrument- this is especially important when analyzing biological sample extracts. Sometimes it is not possible to ascertain the samples are dry with naked eye; an undried sample will cause charging when you hit laser. Ajeet was an expert in drying samples and he has tried out a lot of protocols for obtaining good crystallization of matrix. One of his papers was cited in a recent review where they mention what things must be considered for quantitative MALDI (Wang and Giese, J. Chromatogr. A. 2017, 1486, 35-41).

8.  Pay attention to the method file that is being loaded / used and whether or not it is the correct one. First try out the method with the relevant standards. Don't even spot samples beforehand. Or else, have a second plate with samples and use it ONLY if the standards show up well. Always use the automated acquisition modes for quantitative analysis to remove any user biases. One has to optimize the sample prep, spot drying and acquisition conditions before the automated analysis is employed.

9.  Remember that one can do the analysis either in the reflectron mode or the linear mode; positive or negative mode.

10. Know when “In source decay” could be used beneficially; and when one is better off with CID fragmentation (MS/MS); know the possible “adduct formations” and strategies to tweak them – for example using TFA for improving protonation or using an alkali salt for selective alkali adduct formation

11. We found significant variations when working in the Pune summer, especially when we had to go out with the samples to the central lab. This is less of an issue now that we have the analyzer in our lab; but, keep in mind if you ever have to use the TOF/TOF at CMC. Minimize the transit time, avoid experiments between 10am – 4pm in Apr/May; carry your target plates/samples in an insulated box with ice packs. Increased power load at the lab (NCL) in summer and occasional disruption has happened in the past. When this happens, the ACs go off (even if the instrument has power back up) and the room temperature can go up very rapidly in May leaving us all on the edge. Considering these, as a group we resolved to either try and minimize lengthy quantitative workflows and LC MS  workflows at least in April /May or better still shut down the analyzer. Get enough data before April and focus on write ups; plan vacations in May!! The subsequent months of June – Aug can be a bit testing due to humid monsoons – not as bad an issue as the tropical summer heat; but, do pay attention and watch out!

12. Ajeet says: Oligonucleotides are friendly with Basic MALDI matrices and acidic matrices (CHCA/DHB) causes high fragmentation. HPA is good choice; however if it is not available, try some addition of pyrimidine/pyridine to Sinapinic acid or nicotinic acid. After addition it will not be completely dry but enough to generate ions. In case you have HPA and samples are not drying, place 0.2 uL of acetone on top of matrix+sample layer. It will make homogeneous surface with good crystal. Laser energy optimization is essential in case of oligonucleotide. Laser should not completely ablate the sample from surface (for small molecule, complete ablation is fine).

13. Mass range is important a) ABSciex TOF/TOF:  Reflectron TOF will work better up-to 50kD b) Waters Synapt hybrid MS analyzer: set quadruple (Q1) for high mass range.