Peptide fragmentation by corona discharge induced electrochemical ionization
John R. Lloyd
0
2
Sonja Hess
0
1
0
Address reprint requests to Dr. S. Hess,
California Institute of Technology
, BI 211, MC139-74, Pasadena,
CA 91125, USA
1
Proteome Exploration Laboratory, California Institute of Technology
, Pasadena,
California, USA
2
Proteomics and Mass Spectrometry Facility,
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health
,
Bethesda, Maryland, USA
Fundamental studies have greatly improved our understanding of electrospray, including the underlying electrochemical reactions. Generally regarded as disadvantageous, we have recently shown that corona discharge (CD) can be used as an effective method to create a radical cation species [M] , thus optimizing the electrochemical reactions that occur on the surface of the stainless steel (SS) electrospray capillary tip. This technique is known as CD initiated electrochemical ionization (CD-ECI). Here, we report on the fundamental studies using CD-ECI to induce analytically useful in-source fragmentation of a range of molecules that complex transition metals. Compounds that have been selectively fragmented using CD-ECI include enolate forming phenylglycine containing peptides, glycopeptides, nucleosides, and phosphopeptides. Collision induced dissociation (CID) or other activation techniques were not necessary for CD-ECI fragmentation. A four step mechanism was proposed: (1) complexation using either Fe in the SS capillary tip material or Cu(II) as an offline complexation reagent; (2) electrochemical oxidation of the complexed metal and thus formation of a radical cation (e.g.; Fe e Fe ); (3) radical fragmentation of the complexed compound; (4) electrospray ionization of the fragmented neutrals. Fragmentation patterns resembling b- and y-type ions were observed and allowed the localization of the phosphorylation sites. (J Am Soc Mass Spectrom 2010, 21, 2051-2061) 2010 American Society for Mass Spectrometry
-
emission is thus negligible. In contrast, corona
discharge (CD) is characterized by a stable emission of
blue or red light, depending on the electron densities of
the plasma. CD is a special case in that it oscillates at
steady-state between the Townsend region of the dark
discharge and the glow discharge region, roughly
between 107 A104 A. These oscillations are very well
studied, experimentally by Trichel, hence the name
Trichel pulses, and theoretically by Morrow [7, 8]. In
addition, the flow of electrons out of the electrospray tip
measured in ESI and CD-ESI techniques has been
extensively studied and documented [6, 9]. We have
recently shown that corona discharge (CD) can be used
as an effective method to create a radical cation species
[M] using a regular ESI source and appropriate MS
settings [6]. To achieve a stable CD, the exit tip of a
stainless steel electrospray capillary was extended 3
mm beyond the desolvation gas tube, the nitrogen gas
temperature was raised to at least 500 C, and the ES
high voltage to at least 5 KV. As shown in Figure 1, the
ion plasma created was diverted away from the ESI
spray path by a ground electrode placed behind the ESI
capillary exit. CD at the capillary tip oxidizes the Fe in
the stainless steel, which subsequently oxidizes redox
active analytes when the ionization potential of the
analyte is equal to or lower than that of Fe. Detection of
N-ferrocenyl iodoacetamide (FcIAA) at the low
zeptomolar level was demonstrated. The excellent sensitivity
Sgreatly improved our understanding of
electrosince its inception, fundamental studies have
pray [1]. It is, for instance, now no longer
questioned that the electrospray source also functions as an
electrolytic cell, where electrochemical oxidations can
happen as originally proven by Blades et al. [2] and
extensively studied by van Berkel and Kertesz [3]. The
focus of these studies was the reactions in the gas phase,
and not those at the surface of the electrodes. It is thus
not surprising that until recently, the connection
between the electrolytic nature of electrospray and the
onset of commonly observed discharges (i.e., the release
of electrons) was not well understood. For instance, in a
recent review, Kebarle et al. have associated the onset of
these discharges with cosmic rays or background
radiation. What we have recently shown is that the onset of
the discharges is associated with the electrochemistry
that Kebarle initially described [1, 2]. CD is initiated at
the sharp edge of the capillary due to its high potential
energy and follows the normal voltage-current
characteristics as established by Penning, where dependent on
the current, discharges are classified as dark discharges
(1010 A105 A), glow discharges (105 A ca 1A) and
arc discharges (ca 1A104A) [4 6]. At dark discharges,
very few electrons (1010 A105 A) are released, light
could be attributed to effective electrochemical
ionization and the ability of the enolate form of FcIAA to bind
and accumulate to the Fe in SS capillary [6]. We ter (...truncated)