I concur it could be something in the prep  phase gone wrong. 

Have you changed any lab supply (vials, pipettes, tubes, etc...)recently? Sometimes different supplier could leach out some of their additives. 

A thing seems strange to me, your calibration with cytochrome C is good but an old sample apparently well preserved is not, maybe you could spot some differences in the two processes to restrict the cause?

Did you try cleaning the ion sweep cone and ion transfer capillary and other parts in the source?

Thanks for your responses. We have not attempted to modify the instrumentation yet (other than replacing the column) as samples from other lab groups and supplier standards seem normal. I have to assume that it's a contamination issue with my preparation, though to my knowledge nothing has changed. I will do a complete change of reagents and prepare a new sample in glass to eliminate as many variables as possible.

Dear Clinton,

I am having a similar problem - usually my peptide samples look clean when run on our QE and yield around 1300 IDs, but now (using the same sample prep protocol) I am suddenly seeing late-gradient 1+ contaminants with m/z 376.26 (also 268.14, 536.29, 856.35, and what is possibly trypsin-like adduct at 515.33).

Additionally, there seem to be more 3+ and 4+ ionic species late in the gradient, but the peptides which are detected have very few missed cleavages.

I am at a loss to explain this sudden contaminant problem, as I am always very careful during reagent and sample preparation to avoid contaminants, and have never experienced this problem before.

I thought it might be beneficial to discuss this with you since we have the same 376.26 peak. Have you perhaps found a solution on your side?

Kind regards, Jan

Have you always used diethyl ether?  This is a very strong solvent, and may be leaching something - as others have suggested - from some of your vials or tips, etc.  This was the first red flag for me, although if you've used it successfully in the past it may instead be a red herring.  I did troubleshoot a similar problem once for a colleague, and it turned out to be something in a new batch of scintillation vials.  Once he started rinsing them before using them the problem disappeared.

Thanks again. I've conducted a short experiment by preparing new samples either using my old reagents and handling techniques versus using new reagents and working entirely in either glass or Eppendorf protein Lobind tubes to limit the chance of inappropriate tubes leaching contaminants, particularly due to solvents. I also removed the diethyl ether phase transfer step, relying only on acid precipitation and C18 tips to remove the SDC detergent (I have done this before with success). The number of protein IDs has somewhat improved (to approx. 800 from 500, still short of the 1200 ideal), and the large unexplainable peaks have largely vanished, but I'm still experiencing the strange "fuzzy" regions of the chromatogram as shown in the images above, albeit less severely, where the data acquisition points are not separated. Strangely, this effect was the same in both samples, regardless of my changing the method (no obvious difference between the two sets of reagents/handling methods). Both improved, but are still far short of acceptable. Any suggestions on what might be causing this would be appreciated.

As I mentioned before a dirty ion sweep cone and ion transfer capillary in the source could be the problem. If you are using an LTQ system these parts can easily be accessed and cleaned with out breaking vacuum in the instrument. I was using a triple quad the other day from a different manufacturer and the analyte peak of interest was almost totally absent until I cleaned the desolavtion line (equivalent to the ion transfer capillary) and sampling cone (equivalent to ion sweep cone). After cleaning I got sensitivity down to 10ng/ml of the analyte. Before running this assay which uses 0.1% formic acid in methanol and water, I had used solvents containing 10mM ammonium formate in acetonitrile and water. Apparently the mass spec inlet was fouled with the ion suppressing salts from the prior assay which could not be washed out even after injecting quite a few samples in the new solvent system. 

Thanks Bruce. I have since modified the sample preparation protocol by substituting ethyl acetate for the diethyl ether to remove the detergent. Since doing so the offending large peaks late in the gradient and the "fuzzy" extra peaks throughout the gradient have subsided. The chromatogram appears fairly good at first glance, though overall signal intensity seems oddly low (peaks in the E7 range, despite a loading of about 5ug peptides!) and background is pretty high, nearly 1E6. I would expect the signal intensity to be at least 3-fold higher. Would you expect cleaning the ion sweep cone and transfer capillary to improve signal:noise?

Diethyl ether can also contain the late eluting preservative BHT. Cleaning the source parts should remove any ion suppressing contaminants and restore the sensitivity.

 Hi Clinton,

We are also having the same problem with QE. Could you resolve it? Did cleaning the source help?

@John S Wishnok

you mentioned that rinsing the scintillation vials before using resolved the issue. Rinsing in what? methanol? ACN? We are not using diethyl ether.

Swasti