Phosphorylated proteins span the gamut of protein expression level, from hundreds of millions to a few copies per
cell. However, many of the phosphorylation events associated with canonical cellular signaling pathways occur on proteins expressed at relatively low levels. Since phosphorylation of these proteins is often substoichiometric and transient, phosphopeptides obtained from these proteins after proteolytic digest are nearly impossible to detect in the whole cell lysate or tissue sample, which can generate potentially millions of peptides. Selective enrichment of phosphorylated peptides and proteins is required and has been accomplished in a number of ways, including antiphosphotyrosine antibodies [5
], immobilized metal affinity chromatography (IMAC) [6
], chemical modification, and strong cation exchange chromatography (SCX) [7
Immunoprecipitation (IP) of tyrosine phosphorylated proteins and peptides with high affinity antiphosphotyrosine antibodies [8
] provides good yield and specificity and has been demonstrated on a broad variety of applications [9
]. Several reliable antiphosphotyrosine antibodies are sold commercially. These antibodies primarily recognize phosphotyrosine, but each has some bias toward the surrounding amino acids, and therefore performing the IP with multiple antibodies may increase coverage of the tyrosine phosphoproteome. Since the fraction of tyrosine phosphorylated protein to total protein may vary significantly from sample to sample, experimental optimization of conditions, including relative amount of antibody to total sample protein, is often necessary to reduce nonspecific binding while maximizing yield for the particular sample. It is worth noting that while IP has been succesfully implemented for tyrosine phosphorylation, anecdotal evidence indicates that the analogous pan-specific antibodies against phosphoserine and phosphothreonine tend to be of lower affinity, and therefore yield unsatisfactory enrichment for these subsets of phosphorylated peptides. However, recent work by Matsuoka et al.
] has demonstrated the potential of using multiple phosphospecific antibodies recognizing ATM/ATR substrate phosphorylation sites to identify and quantify hundreds of serine and threonine phosphorylation sites matching the ATM/ATR kinase motif. Since many phospho-specific antibodies have off-target affinity, it may be that this strategy could be applied to a variety of serine/threonine kinases, effectively supplementing the need for high affinity pan-specific phospho-serine/threonine antibodies, and enabling network analysis of serine/threonine phosphorylation, one motif at a time.
For many applications, the goal is to generate a global view of serine, threonine, and tyrosine phosphorylation within the sample rather than focusing specifically on a selected subset of phosphorylated peptides. Perhaps the most common technique to enrich for global phosphorylation is IMAC, which is based on the high affinity of phosphate groups for metal ions such as Fe3+
One of the main limitations associated with IMAC-based phosphopeptide enrichment has been the nonspecific retention of nonphosphorylated acidic peptides, due to the weak affinity between negatively charged carboxylates and positively charged metal ions. However, conversion of carboxylate groups to esters effectively eliminates nonspecific retention of nonphosphorylated peptides on the IMAC column [14
]. This method has also been used in an automated platform involving online IMAC, nano-LC, and ESI-MS, enabling reproducible detection and identification of phosphopeptides in a low-femtomole range [15
], and may be coupled with a stable-isotope labeling step for relative quantification [14
]. Since different metal ions appear to enrich for slightly different subsets of phosphorylated peptides, maximal coverage of the phosphoproteome may be obtained by multiple analyses with different metals, or by mixing multiple metal ions in a single IMAC enrichment step.
Within the past couple of years, titanium dioxide (TiO2
) has emerged as the most common of the metal oxide affinity chromatography (MOAC)-based phosphopeptide enrichment methods. This technique requires significantly shorter preparation time and offers increased capacity relative to IMAC resins with the same bed volume. Since this method exploits the same principle as IMAC, it is similarly prone to nonspecific retention of acidic nonphosphorylated peptides. However, loading peptides in 2,5-dihydroxybenzoic acid has been shown to reduce nonspecific binding to TiO2
, thereby improving phosphopeptide enrichment without chemical modification of the sample [16
]. Overall, TiO2
is often considered to be interchangeable with IMAC, in that similar sample levels (e.g.
, micrograms of protein) can be analyzed and hundreds of sites per
sample can be identified when either technique is used as the sole enrichment method, although each method has demonstrated differential bias and selectivity.
As an alternative to metal-ion-based enrichment strategies, SCX has been successfully used to separate phosphorylated peptides from peptide mixtures for subsequent MS analysis [7
]. In this technique, binding to the SCX column is dependent on columbic interaction between negatively charged resin and positively charged peptides. If sample loading is performed under strong acidic conditions (pH ~2.7), carboxylates are rendered neutral, while the phosphate group retains a negative charge. As a result, the total charge of phosphorylated tryptic peptides is reduced from + 2 to + 1, and the interaction strength with the SCX resin is correspondingly reduced. Elution with a gradient of increasing salt concentration thus allows phosphopeptides to elute earlier relative to nonphosphorylated peptides, providing semiselective enrichment [7
]. To reduce the nonphosphopeptide background, a second, IMAC-based enrichment step has been performed on SCX fractions, enabling the identification of thousands of phosphorylated peptides from given samples [7
]. As another variation and improvement of the SCX method, a mixed-bed resin comprised of a blend of anion and cation exchangers (ACE) has been recently proposed for phosphopeptide enrichment, increasing retention of acidic peptides, and reducing retention of basic and neutral peptides by the added anion-exchange resin, which in turn improved the identifications of phosphopeptides by 94% over SCX [19
Phosphorylation enrichment by SCX-based fractionation, either solely or coupled with other enrichment steps, has successfully been applied to identify large numbers of phosphorylation sites (in the order of thousands). However, it is worth noting that the technique, as implemented to date, requires a large amount of starting material (tens of milligrams of protein) which makes it inapplicable to samples that are available in small or limited quantity. In addition, SCX fractionation decreases the complexity of the starting samples by dividing it into many fractions, each of which requires a separate MS analysis, leading to the possibility of up to 100 MS analyses for each biological replicate. The sample requirements, analysis time, and labor associated with each biological sample has unfortunately limited the application of this technique such that few studies have incorporated biological replicates.
Several laboratories have taken the approach of chemically modifying the phosphate to provide an affinity enrichment tag. For instance, the phosphate groups on serine and threonine can be removed by β-elimination and replaced by ethanedithiol coupled to a biotin tag, making it possible to purify modified peptides using an avidin affinity column [20
]. The primary disadvantage of this approach is that tyrosine phosphorylation does not undergo β-elimination, and therefore these peptides are not enriched by this method. It is also possible to directly attach an affinity tag to the phosphate through phosphoramidate chemistry (PAC). Recent improvements in this approach have improved the yield by reducing the number of steps, making the approach much more user-friendly [21
Different enrichment methods may yield different pools of phosphopeptides from the same peptide mixture, as recently shown in a comparative study conducted by the Aebersold group, where PAC, IMAC, and two types of TiO2
methods were employed to isolate phosphopeptides from a tryptic digest of Drosophila melanogaster
Kc167 cells [22
]. Performing multiple analyses with several complementary phosphopeptide enrichment methods may be the best way to maximize depth of coverage, albeit at the cost of increased sample consumption and reduced throughput.
It is often the case that any single enrichment step does not provide sufficient specificity when dealing with complex biological samples. Therefore, double enrichment, as in the above scenario with IMAC and SCX, is often required to improve phosphopeptide analysis. In another example, our laboratory has combined antiphosphotyrosine peptide IP with IMAC to analyze tyrosine phosphorylation in murine adipocytes [23
], human Jurkat cells [24
], and in the epidermal growth factor receptor (EGFR) signaling network in human mammary epithelial cells (HMECs) [25