Common errors in mass spectrometry-based analysis of post-translational modifications

Abstract

Mass spectrometry (MS) is a powerful tool to analyze complex mixtures of proteins in a high-throughput fashion. Proteome analysis has already become a routine task in biomedical research with the emergence of proteomics core facilities in most research institutions. Post-translational modifications (PTMs) represent a mechanism by which complex biological processes are orchestrated dynamically at the systems level. MS is rapidly becoming popular to discover new modifications and novel sites of known PTMs, revolutionizing the current understanding of diverse signaling pathways and biological processes. However, MS-based analysis of PTMs has its own caveats and pitfalls that can lead to erroneous conclusions. Here, we review the most common errors in MS-based PTM analyses with the goal of adopting strategies that maximize correct interpretation in the context of biological questions that are being addressed. Finally, we provide suggestions that should help mass spectrometrists, bioinformaticians and biologists to perform and interpret MS-based PTM analyses more accurately.

Keywords: Immonium; Localization; PTM; SUMO; Signature; Technology; Ubiquitin.

Figure 1

Ability of mass spectrometers to distinguish PTMs. At different levels of mass accuracy of the mass spectrometer used, the number of modifications that are uniquely identifiable varies. For example, an ion-trap mass spectrometer which has ~100 ppm of mass accuracy would not be able to uniquely distinguish almost half the modifications that are annotated in the UniMod database while a mass spectrometer with a 1 ppm mass accuracy can distinguish most of them.

Figure 2

Homologous domain derived peptides are shared across many proteins. (A) Domain structure for Src family nonreceptor tyrosine kinases is shown at the top. The bottom part shows an alignment of tyrosine kinase domains of five murine members of the Src family of tyrosine kinases. The peptide sequences highlighted in red (LIEDNEYTAR or IIEDNEYTAR) are isobaric and shared by the indicated kinases. (B) A peptide sequence (I/LIEDNEYTAR) was observed to be highly phosphorylated at its tyrosine residue. However, this sequence can be derived from any of the five tyrosine kinases shown in Panel A, making it impossible to deduce the identity of the particular tyrosine kinase that is phosphorylated in the sample analyzed. Two fragment ions (y3 and y4) clearly indicate tyrosine phosphorylation.

Figure 3

Incorrect assignment of a di-Glycine modification. A peptide sequence (NIPIALCTSSNKTK) from a hypothetical protein (YKL033W-A) of Saccharomyces cerevisiae was identified with the di-Glycine modification which serves as a signature for ubiquitylation. In the sequence, there are two lysine residues: one terminal and one internal. MaxQuant indicated a higher probability of terminal lysine being the site of ubiquitylation, but a careful manual validation revealed two signature fragment ions (y1 and y2, marked in red) indicating that the actual ubiquitylation site is likely to be the internal lysine residue instead. Note that lysine was labeled with heavy lysine (¹³C₆¹⁵N₂)for a quantitative proteomic analysis.

Figure 4

Co-fragmentation of peptides bearing a single phosphate but on two different tyrosine residues. Two different tyrosine phosphorylated peptides (YYEGYYAAGPGYGGR and YYEGYYAAGPGYGGR, the site of phosphorylation is underlined) were sampled in one MS/MS scan due to co-fragmentation. In this case, the probability of phosphorylation localization on either site is less than 0.75 which is used as a general a cutoff. However, in this case, both of these modified sites will be missed.

Figure 5

Inaccurate quantitation of co-eluting isobaric peptides. Two signature fragment ions (y3 and y4) clearly distinguish the two isobaric phosphopeptides bearing the phosphate on two different tyrosine residues. However, the isobaric phosphopeptides elute together on liquid chromatography, likely leading to inaccurate quantitation of both phosphopeptides.

Figure 6

Multiple explanations of a quantitative value measured by MS. Shown in Control are two copies of modified peptide sequence and one copy of unmodified counter peptide sequence in control. After a perturbation, modified peptide was reduced to a half of Control (i.e. only one copy in Experiment). There are four possible scenarios that could explain the observation by MS: number of modified peptides was reduced by a half while total number of peptides remains unchanged (case 1) or altered (case 2). A modified peptide in control has obtained an additional modification(s) of the same (case 3) or a different kind (case 4). All four cases resulted in the identical result in MS.