you may try to calibrate the mass directly before applying your sample 

Hi Waheed, I'm not an expert for MS/MS but loss of signal sensitivity can have different reasons. 1. Your derivatized sample show no ionization. 2. Your derivatized sample is outside of the scan range of the ms. 3. Your derivatized sample doesn't eluate from your column (have a look on the system pressure). 4. Your derivatized sample shows interferences with the sample matrix. Maybe this literature site will be helpful to you: http://pubs.acs.org/doi/abs/10.1021/ac4015116 Greetings Markus

I think Markus Christ  covered almost all the possibilities. For your particular case,  I would be more focused on the ionization scheme, and the matrix effects, since you still see your  sample of interest eluting through the column and being detected.

Washeed,

Maybe the bisphosphonate alters the ionization efficiency in positive mode, so you lose sensitivity. There are four sites in the Alendronate that can lose protons and make negative ions. I assume that when the conjugate is made it will use up one of those sites, but it still leaves three sites that can lose a proton to make a negative ion. This may reduce the ability to make a positive ion by proton attachment. You might consider running in negative ion mode, preferably at a higher pH than you might ordinarily use. If you insist on running in positive mode then consider using a lower pH than normal.

Hi waheed,

I agree with Alan and Markus.

I feel that the ionization of you modified peptide would definitely be reduced in positive mode because A.) one NH2 is taken away, so one less positive charge and then, because a biphosphonate is added, it losses two more positive charge B.) This is a long peptide, which means you are probably not detecting molecular ion but rather higher charged species (z=2,3,4 etc). The peak for the peptide would then show up at a lower m/z. When you modify the peptide and reduce +3 charge, it becomes difficult for this long chain to acquire higher charges and thus bulk of your analyte ionises at m/z beyond the detection limit of you MS (2000 is the typical limit on m/z of usual LC-MS/MS used, but most accuracy and sensitivity is between ~700-1200 m/z)

My quick calculation says that you peptide's molecular weight is 3972. Once modified the molecular weight would be ~4200. A peptide of this molecular weight needs to be atleast quadruple charged to bring the m/z down to most sensitive regime (z=4, m/z= 4200/4 =1050). You might detect something at z=3 (m/3= 1400) but that wont be the most sensitive.

Simply put, your modified peptide is not able to acquire that much charge before it hits the detector.

Thank you everyone for all the information and suggestions that have cleared my path I will try on some what is being suggested and see if its even feasible to take on this project on LCMS or not. Once again appreciate everyone's input. Thank you.