Exploring the Fragmentation of Phosphopeptides from AP MALDI QqLIT in both Positive and Negative Polarities

• Phosphate loss from the fragmentation of phosphopeptide ions was studied using an AP MALDI source coupled to a hybrid triple-quadrupole linear ion trap (QqLIT) mass spectrometer
  
  
• Neutral loss of 98 Da, corresponding to H₃PO₄, for phosphotyrosine-containing peptides was verified to be dependent on the presence of Lys and Arg in the positive polarity (in agreement with Moyer et al.1), but independent of amino acid structure in the negative polarity
  
  
• Fragmentation of phosphopeptides to yield -79 Da, corresponding to PO3-, was found to be dependent on the collision energy applied and the mass being fragmented
  
  
• A robust method to identify phosphopeptides using NL scanning for 98 Da in the negative ion mode was demonstrated

• Protein phosphorylation is an important post-translational modification that occurs in biological pathways
  
  
• MALDI mass spectrometry has been a popular method for phosphopeptide analysis
  
  
• Moyer et al.¹ showed that AP MALDI – MS/MS in positive ion mode for singly-charged phosphopeptides containing phosphoserine and phosphothreonine resulted in neutral loss of 98 Da, while phosphotyrosine yielded a neutral loss depending on the presence of arginine or lysine in the sequence
  
  
• This work investigated the fragmentation of singly-charged phosphopeptides in both positive and negative ion modes, using an AP MALDI – QqLIT combination
  
  
• Objective was to better understand MALDI MS/MS fragmentation of phosphopeptides so that a robust method for phosphopeptide ID could be developed

Instrumentation

• A hybrid QqLIT (4000 Q TRAP^®; system) from AB/MDS SCIEX was combined with a MassTech AP MALDI Pulsed Dynamic Focusing source (AP/MALDI-PDF Model 712)

FIGURE 1. Schematic of AP MALDI PDF on a hybrid triple quadrupole – linear ion trap MS.

• Fragmentation was studied using the high sensitivity LIT mode of the 4000 Q TRAP^® system
  
  
• Potential markers for phosphopeptide ID were studied, and included: 1) neutral loss of 98 Da in both polarities, 2) product ion of +216 Da (immonium ion), and 3) product ion -79 Da (phosphate ion)

• Precursor ion (PI) and neutral loss (NL) triple quadrupole scanning modes were applied to the potential phosphopeptide markers, and used to screen complex samples for phosphopeptides

Materials and Sample Preparation

Samples:

• -casein (Sigma Chemical Co., St. Louis, MO): Digested with trypsin (ratio of 20:1 protein:trypsin) for 4 hours at 37°C
  
  
• Synthetic phosphopeptides: MW: 748.7 Da (M-2170), 1128.1 Da (M-2035) without Lys or Arg (BaChem). MW: 1333 Da (#24441) contained Lys, and 1630 Da (#24544) contained both Lys and Arg (AnaSpec). Phosphopeptides were reconstituted in 0.1% TFA

MALDI Preparation:

• -cyano-4-hydroxycinnamic acid (-cyano, Sigma)
  
  
• 2,5-dihydroxy benzoic acid (DHB, Fluka)

dissolved in 70% ACN/
30% H₂O with 0.1%TFA
(Sigma)

• 1 µL, standard dried-droplet method on stainless steel plates (V700666, AB)

Phosphopeptide Fragmentation Study:

• Spectra in Figs. 2-5 show that for peptides containing pTyr without Arg or Lys, no NL of 98 Da occurs in the positive ion mode but does occur in the negative ion mode
  
  
• Increasing the collision energy (CE) shifts product fragments to lower m/z
  
  
• Fragmentation is dependent on collision energy and mass, where low mass ions require low CE to fragment and high mass ions require high CE to fragment
  
  
• Same results were found with DHB, data not shown
  
  
• Candidate phosphopeptide markers are: NL (-98 Da), and PI (+216 Da)

Applying triple-quadrupole scans to phosphopeptide mixtures:

• Figure 6 shows that NL (-98 Da) scanning can specifically detect phosphopeptides from a mixture of digest peptides
  
  
• In contrast, PI (+216 Da) is less effective for phosphopeptide ID when analyzing a mixture of peptides

• AP MALDI MS/MS fragmentation of phosphopeptides revealed that neutral loss of 98 Da was not dependent on peptide composition in the negative ion mode
  
  
• An AP MALDI triple-quad method using scanning of NL (-98 Da) demonstrated the ability of the method to pick out phosphopeptides from complex mixtures
  
  
• Future work will explore the application of the technique to real biological samples

¹Moyer, SC; Cotter, RJ; Woods, AS. J. Am. Soc. Mass Spectrom. 2002, 13, 274-283.

ACKNOWLEDGMENTS: The authors thank Deolinda Fernandes for the preparation of the -casein digest.