Hi,

The first and last spectra attached seem to mainly contain background signals, but the other three contain protein-like signals around 11 and 14 kDa.

Can you provide some additional information on the protein(s) you're studying?

And why do you seem to be using two different matrices?

Thanks!

Hi Sobia Ejaz,

I go through the images. All are different showing different. I agree with Dirksen. Its very simple to know mass of proteins by MALDI TOF. Please find the theoretical value (TV)of your interest protein(based on amino acids). Now compare TV with your experiment MALDI-TOF.

1. Did you purified your protein by FPLC?

2. MALDI-TOF is very sensitive method so you cannot do without purification.

3. If your protein have high molar salt solution will you get extra peaks.

4. can you try with different ratio of matrix and your protein ?

5. MALDI TOF very sensitive so you have to try 2-3 time to confirm . I worked more than 8 months for 3 proteins, can you believe. I know the struggle pain.

This may help to you need any information and suggestion feel free ask me.

Thank you

With regards

Vikky R

What were you expecting to see in these spectra? A clean spectrum with only one peak with good shape and resolution, say at 14.1 kDa for example?

How did you calibrate your instrument? You must have used a mixture of 'pure' proteins with known sequence and theoretical molecular masses, accounting for any modifications - like known number of disulfides in insulin.

Are the peaks in the spectrum used for calibration show good resolution and peak shape? Are there lot of unexplained peaks in the calibration spectrum? Are the peaks too broad at the base or have lot of tailing on the higher mass side?

Once you are convinced that your calibrants are 'good', and the instrumental parameters are optimized for resolution and sensitivity, then you have an idea of what kind of spectrum you should get for a clean sample of a protein without molecular heterogeneity.

Sample history is very important - Did you purify the protein from natural sources? Or is this a recombinant protein? Are there any post-translational modifications you are expecting to see?

Without such background about the sample and the instrument used, etc. it is difficult to help with any interpretation of the data.

Thank you dear for your response. Actually I extracted my sample from a medicinal plant and then ran its SEC on Sephadex G-50 as I need to work on low molecular weight proteins and then purified using HPLC C4 column and then analysed through MALDI-MS in order to determine its molecular weight. But along with the molecular weight peak, there are also abundant ion peak before that peak. I just want to know what are these peaks.

Thank you for your gentle support.

Hi,

Based on the data provided, it is impossible to determine what the peaks observed at lower mass represent. It seems your protein of interest is 14.6 kDa and in some spectra you observe the doubly charged form at 7.3 kDa. Other than that, the species detected can be peptides, or other compounds extracted from your medicinal plant.

You could try to run LC-MS/MS, maybe also of a digested sample, to see what other peptides/proteins are in you sample, as LC-MS/MS allows for peptide sequencing and, potentially, protein identification.

Good luck!

Thank you dear.......