UV peak but no TIC peak for a protein - why?

[biochemxx]

We are using reverse phase HPLC coupled to ESI-TOFMS for the analysis of intact protein mass. We are running a protein standard that contains 4 proteins and we are currently injecting 10ug of each protein. There are very nice, strong, seperated UV peaks for each protein, and good TIC peaks for 3 of the proteins, but one of them is not showing up at all on the TIC. The one that is not showing up is holotransferrin, it is the largest protein (77kDa vs 10-20kDa for the other proteins).

Is the protein not ionizing or something? What can we do to make it show up on the TIC?

More info:
Column is a 4.6mm 300A C18
Mobile phase A is 0.1% TFA (we have also tried formic acid)
B is acetonitrile with 0.1% TFA
Temp is 40C
Mass analyzer is an Agilent 6200 series TOF

[Karen01]

We are using reverse phase HPLC coupled to ESI-TOFMS for the analysis of intact protein mass. We are running a protein standard that contains 4 proteins and we are currently injecting 10ug of each protein. There are very nice, strong, seperated UV peaks for each protein, and good TIC peaks for 3 of the proteins, but one of them is not showing up at all on the TIC. The one that is not showing up is holotransferrin, it is the largest protein (77kDa vs 10-20kDa for the other proteins).

Is the protein not ionizing or something? What can we do to make it show up on the TIC?

More likely it's not flying.... try increasing cone voltage after the smaller proteins elute (to avoid fragmenting them)

- Karen

[Jetjamnong]

Mobile phase A is 0.1% TFA (we have also tried formic acid)
B is acetonitrile with 0.1% TFA

...TFA (trifluoroacetic acid) is a commonly used mobile phase additive for reversed-phase HPLC (RP-HPLC) separations of proteins and peptides. However, TFA interferes with and significantly reduces the LC/MS signal, lowering sensitivity. The ideal column for modern RP-LC/MS analysis should provide symmetrical peak shape without TFA in the mobile phase. The
highly inert surface of Discovery BIO silica results in columns that give symmetrical and efficient peaks for
peptides without TFA for maximum LC/MS sensitivity. A major challenge facing biotechnology and proteomics researchers
and others working with peptides or peptide maps is the need to detect and identify single peptides often at very low concentrations in extremely complex samples. Liquid chromatography coupled with mass spectroscopy (LC/MS) has become an invaluable tool to meet this challenge. Because of its high resolving power, RP-HPLC is the preferred separation mode for peptides. Traditionally, TFA is used in the mobile phases for RP-HPLC peptide separations. Ionic mobile phase additives like TFA serve one or more of the following functions: pH control (buffering), complexation with oppositely charged ionic groups to enhance RP retention (ion pairing), or suppression of adverse ionic interactions between peptides and silanol groups on the silica. The latter function is necessary when using RP-HPLC phases with high silanol activity. While TFA has little effect on UV detection, it has serious disadvantages for LC/MS detection. First, typical concentrations of TFA (0.1% v/v) have high surface tension and prevent efficient spray formation (nebulization). Second, TFA ions in the gas phase ion-pair with the peptide’s basic groups suppressing their ionization and reducing sensitivity.
Without TFA, the MS is able to detect much lower concentrations of these peptides. An added benefit is that at low TFA concentrations, resolution is improved because small differences in peptide retention are not masked. At 0.1% TFA, they co-elute. Therefore, from the mobile phase standpoint, the best LC/MS method employs ionic additives other than TFA that are still volatile, can provide pH control, and do not strongly ion-pair with the peptides...

Resource : http://www.sigmaaldrich.com/Graphics/Su ... /11547.pdf