Multidimensional Separations for Deep Coverage of the Phosphoproteome
Despite the availability of highly selective phosphopeptide enrichment methods and mass spectrometers with ever-increasing data acquisition rates, the complexity and dynamic range of the cellular phosphoproteome are still far too great to permit its full characterization by direct mass spectrometric analysis. Fractionation of the phosphoproteome, typically at the peptide level, using multidimensional chromatographic separations, is a necessary prerequisite for obtaining in-depth, comprehensive coverage (see Figure 2.7). Fractionation in itself is generally not sufficient to permit efficient phosphopeptide identifications from a complex mixture; rather it should be paired with a selective enrichment step. While this fractionation may occur prior to, or following the affinity enrichment, we  and others  have shown that fractionation prior to enrichment results in better selectivity in the enrichment and overall more in-depth coverage of the phosphoproteome. Phosphoproteomics prefractionation is typically performed offline to accommodate the processing of up to several milligrams of protein lysate required for large-scale, in-depth studies. Less frequently, a multidimensional separation or phosphopeptide enrichment step is fully integrated online with the mass spectrometer [224-226]. Ideally, any multidimensional separation strategy combines a chromatographic mode of fractionation highly orthogonal to the final RP separation interfaced to the MS.
Multidimensional chromatographic separations for phosphoproteomics are invariably based on exploiting the unique physicochemical properties with which the phosphorylated amino acid residue bears on modified peptides: (i) the ability of the phosphate group as a weak acid to carry up to two negative
charges at physiological pH (pKa1 2.1, pKa2 7.2)  and (ii) the polarity of the phosphoamino acid ester, which imparts a strongly hydrophilic character on phosphopeptides relative to their nonphosphorylated counterparts .
Chromatographic enrichment methods based on ion-exchange mechanisms are widely utilized due to the difference in solution charge states between phosphorylated and nonphosphorylated peptides. In strong cation-exchange (SCX) chromatography, peptides are bound to a column containing a hydrophilic, anionic stationary phase through their positively charged functional groups. Phosphopeptide enrichment using SCX is typically conducted at low pH (2.7). Under these conditions, the N-terminal amino group as well as the side chains of arginine and lysine will be protonated. Thus, a proteotypic tryptic peptide has a net charge of +2. Addition of a negatively charged phosphate group to the peptide will reduce the charge by one, providing a mechanism for separation and enrichment of phosphopeptides from the bulk of nonphosphorylated peptides . Though widely applied, it is now well known that many phosphopeptides do not elute in the 1+ fractions. Peptides containing greater than one phosphate can carry a net zero or negative charge and will elute with highly acidic nonphosphorylated peptides in the unbound fraction; this negatively impacts subsequent IMAC and MOAC binding selectivity. Other peptides that are charge reduced, including peptides derived from protein N-termini (N-acetylated) and C-termini (lacking Arg or Lys), will coelute with the +1 phosphopeptide pool. Additionally, phosphopeptides containing histidine residues, or missed tryptic cleavages introducing internal Lys or Arg residues, will also not be found in the early phosphopeptide-rich fractions. Finally, SCX is a fairly low-resolution technique and is only moderately orthogonal to reverse phase as a component of a 2D strategy . Despite these considerations, SCX has been widely used as a first-dimension separation coupled with either IMAC [119, 188] or MOAC [5, 230] enrichment for large-scale phos- phoproteomics studies, though it is now recognized that fractions should be taken across the entire SCX gradient to provide the greatest depth of coverage.
Strong anion-exchange (SAX) chromatography separation relies on the binding of negatively charged peptides to a positively charged stationary phase. In contrast to SCX, under neutral to mildly acidic binding conditions, the strongly acidic phosphopeptides show greater retention on SAX than nonphosphoryl- ated counterparts, which predominate in the early eluting fractions. Using SAX, partial fractionation of the phosphoproteome based on number of phosphates is observed . SAX chromatography has been increasingly applied to phosphopeptide fractionation [232-234].
HILIC is a high-resolution separation technique where the primary interaction between a peptide and the neutral, hydrophilic stationary phase is hydrogen bonding. In HILIC, retention increases with increasing polarity (hydrophilicity) of the peptide, opposite to the trends observed in RP . HILIC has the highest degree of orthogonality to RP of all commonly used
peptide separation modes , making it the ideal first-dimension separation for peptide-based proteomics. Samples are loaded at high organic solvent concentration and eluted by increasing the polarity of the mobile phase (e.g., an inverse gradient of ACN in water). Under these conditions, phosphopeptides are strongly retained because phosphoamino acids are far more hydrophilic than the twenty standard amino acids. Using an optimized gradient, phospho- peptides can be partitioned away from the bulk of nonphosphorylated peptides and further fractionated based on their hydrophobicity and the number of phosphorylated residues . This greatly improves the selectivity of subsequent chemoaffinity enrichment techniques. An advantage of HILIC is that the volatile, salt-free TFA/ACN buffer system employed makes the fractions compatible with direct IMAC or MOAC capture. HILIC has been frequently applied in multidimensional phosphopeptide isolation workflows [33, 235-237].
Electrostatic repulsion hydrophilic interaction chromatography (ERLIC) is a technique closely related to HILIC. Also known as ion-pair normal phase, ERLIC employs HILIC on a weak anion-exchange (WAX) column . Performing separations at low pH, acidic side chains and C-terminal carboxy groups are protonated, thus reducing their effect on retention. Basic side chains and N-terminal amines are positively charged and electrostatically repulsed by the column. The negatively charged phosphate group interacts favorably with the WAX column, leading to increased retention time. Separation is performed in high organic solvent, which superimposes a hydrophilic interaction mode on the electrostatic effects and further promotes interaction of the phosphate group with the stationary phase, such that phos- phopeptides are separated from the bulk of the nonphosphorylated peptides . However, since nearly half of the phosphopeptide-containing fractions also contain 50% or more nonphosphorylated peptides , it would seem obvious that to make the most efficient use of the MS and provide deeper coverage of the phosphoproteome, these fractions would benefit from further enrichment by IMAC or MOAC. Unfortunately, the separation of phospho- peptides by ERLIC requires 20 mM sodium methylphosphonate buffer, which would need to be removed prior to further enrichment or direct MS analysis. While not in widespread use, ERLIC has been successfully employed in a number of phosphoproteomics studies [240-242].
High-pH (HpH) reversed phase is a separation mode that is gaining ground as the first-dimension fractionation for in-depth phosphoproteome studies. Intuitively, it would seem that a multidimensional separation strategy employing two successive rounds of RP chromatography would provide only marginal improvements in practical 2D peak capacity over a single dimension of RP separation and would be inferior to all 2D separations based on different physicochemical properties (i.e., charge or hydrophilicity). However, systematic evaluation of RP-RP 2D HPLC conditions revealed that while altering ion-pairing agents and stationary phases yielded only subtle changes in orthogonality, a change in the pH of mobile phase had a more pronounced effect on peptide retention behavior . This is attributed to differences in individual peptide isoelectric (pI) points, which range widely from 3 to 12 and are dependent on the number and composition of ionizable termini and side chain functionalities. Acidic peptides (pI < 5.5) are more strongly retained at pH 2.6, when the carboxylic moieties are not ionized, in contrast to basic peptides (pI > 7.5), which are more strongly retained under pH 10 conditions. Separations are routinely performed at ~pH 10 in ammonium formate- or ammonium hydroxide-containing buffers, though it has been observed that formate buffers can degrade column performance . Despite RP possessing the highest resolution of routine chromatographic separation modes, even under optimal conditions, differential pH-based RP-RP separations are only semiorthogonal. To further improve orthogonality, a concatenation strategy was introduced in which systematic pooling of the early, middle, and late fractions is performed prior to analysis by low-pH RPLC-MS . Several recent examples have shown this to be a highly effective first-dimension separation, performed either prior to  or following phosphopeptide affinity enrichment [134, 234]. Direct comparison would suggest that for phosphoproteom- ics analysis, HpH is superior to SCX, particularly with respect to the fractionation of singly phosphorylated peptides . It is thus likely that HpH will eventually supplant SCX as a primary first-dimension separation mode.