immobilized metal affinity chromatography (IMAC) and metal
oxide chromatography (MOC)) is used for their effective MS analysis [5, 6]. The combined efficient phosphopeptide enrichment
with advanced LC-MS platforms enables routine identification of
more than 10,000 phosphorylation sites in a single LC-MS/MS
analysis (or 50,000 sites with multidimensional separation
approaches) [7–12]. Despite these advances, significant gaps in
phosphoproteome coverage still exist, especially for small, hydrophilic phosphopeptides with low abundance. The use of trypsin, the
protease of choice for most shotgun proteomics studies [13], generates peptides well suited for ESI-MS [14]. However, some peptides containing hydrophilic phosphate groups can become too
hydrophilic to be retained by the widely utilized reversed-phase
(RP) LC columns for separation of peptide mixtures prior to
MS. Therefore, current MS-based phosphoproteomics platforms
may inadvertently overlook certain portions of the highly hydrophilic phosphoproteome.
We recently developed a simple, easily implemented method to
introduce a commonly used tandem mass tag (TMT) to increase
peptide hydrophobicity, effectively enhancing RPLC-MS analysis of
hydrophilic phosphopeptides [15]. TMT reagents are broadly used
in shotgun proteomics for multiplexed sample analysis by labeling
primary amines (N-terminus and ε-amine group of lysine) with
chemical tags through N-hydroxysuccinimide (NHS) chemistry.
Different from conventional TMT labeling, this TMT-assisted
method capitalizes on TMT labeling occurring before C18-based
solid phase extraction (SPE) to avoid the loss of hydrophilic phosphopeptides during C18 SPE cleanup and uses a no-primary amine
buffer for sample preparation so that TMT reagents can be effectively labeled on peptides before C18 SPE (Fig. 1). In this chapter,
we describe in detail a protocol for the TMT-assisted method that
can be used for convenient quantitative phosphoproteome profiling
of very hydrophilic phosphopeptides. We highlight critical steps in
sample preparation for phosphoproteome analysis and briefly
describe the analysis of phosphoproteomics data using MaxQuant
[16, 17] and demonstrate how the protocol is appropriate for
global analysis of the hydrophilic phosphoproteome and rescuing
highly hydrophilic phosphopeptides with important biological
functions.
We systematically evaluated the effect of the TMT-based
method on global phosphoproteomics analysis. Using equal
amounts of tryptic peptides from MCF-7 cell lysates with or without TMT0 labeling, we demonstrated that the number of identified
phosphopeptides with TMT0 labeling (N ¼ 10,311) was comparable to that without TMT0 labeling (N ¼ 11,256) (Fig. 2a). A total
of 16,104 unique phosphopeptides were identified from the two
sets of samples with 5463 (34%) phosphopeptides in common
(Fig. 2b), an overlap that is significantly lower than the typical
248
Chia-Feng Tsai et al.
oxide chromatography (MOC)) is used for their effective MS analysis [5, 6]. The combined efficient phosphopeptide enrichment
with advanced LC-MS platforms enables routine identification of
more than 10,000 phosphorylation sites in a single LC-MS/MS
analysis (or 50,000 sites with multidimensional separation
approaches) [7–12]. Despite these advances, significant gaps in
phosphoproteome coverage still exist, especially for small, hydrophilic phosphopeptides with low abundance. The use of trypsin, the
protease of choice for most shotgun proteomics studies [13], generates peptides well suited for ESI-MS [14]. However, some peptides containing hydrophilic phosphate groups can become too
hydrophilic to be retained by the widely utilized reversed-phase
(RP) LC columns for separation of peptide mixtures prior to
MS. Therefore, current MS-based phosphoproteomics platforms
may inadvertently overlook certain portions of the highly hydrophilic phosphoproteome.
We recently developed a simple, easily implemented method to
introduce a commonly used tandem mass tag (TMT) to increase
peptide hydrophobicity, effectively enhancing RPLC-MS analysis of
hydrophilic phosphopeptides [15]. TMT reagents are broadly used
in shotgun proteomics for multiplexed sample analysis by labeling
primary amines (N-terminus and ε-amine group of lysine) with
chemical tags through N-hydroxysuccinimide (NHS) chemistry.
Different from conventional TMT labeling, this TMT-assisted
method capitalizes on TMT labeling occurring before C18-based
solid phase extraction (SPE) to avoid the loss of hydrophilic phosphopeptides during C18 SPE cleanup and uses a no-primary amine
buffer for sample preparation so that TMT reagents can be effectively labeled on peptides before C18 SPE (Fig. 1). In this chapter,
we describe in detail a protocol for the TMT-assisted method that
can be used for convenient quantitative phosphoproteome profiling
of very hydrophilic phosphopeptides. We highlight critical steps in
sample preparation for phosphoproteome analysis and briefly
describe the analysis of phosphoproteomics data using MaxQuant
[16, 17] and demonstrate how the protocol is appropriate for
global analysis of the hydrophilic phosphoproteome and rescuing
highly hydrophilic phosphopeptides with important biological
functions.
We systematically evaluated the effect of the TMT-based
method on global phosphoproteomics analysis. Using equal
amounts of tryptic peptides from MCF-7 cell lysates with or without TMT0 labeling, we demonstrated that the number of identified
phosphopeptides with TMT0 labeling (N ¼ 10,311) was comparable to that without TMT0 labeling (N ¼ 11,256) (Fig. 2a). A total
of 16,104 unique phosphopeptides were identified from the two
sets of samples with 5463 (34%) phosphopeptides in common
(Fig. 2b), an overlap that is significantly lower than the typical
248
Chia-Feng Tsai et al.